Biophysical investigation of type A PutAs reveals a...

Biophysical investigation of type A PutAs reveals aconserved core oligomeric structureDavid A. Korasick1, Harkewal Singh2, Travis A. Pemberton2, Min Luo2, Richa Dhatwalia2 andJohn J. Tanner1,2

1 Department of Biochemistry, University of Missouri, Columbia, MO, USA

2 Department of Chemistry, University of Missouri, Columbia, MO, USA

Keywords

analytical ultracentrifugation; oligomerization;

proline catabolism; small-angle X-ray

scattering; X-ray crystallography

Correspondence

J. J. Tanner, Department of Biochemistry,

University of Missouri, Columbia, MO

65211, USA

Fax: +1 573 882 5635

Tel: +1 573 884 1280

E-mail: [email protected]

(Received 15 February 2017, revised 6 June

2017, accepted 12 July 2017)

doi:10.1111/febs.14165

Many enzymes form homooligomers, yet the functional significance of self-

association is seldom obvious. Herein, we examine the connection between

oligomerization and catalytic function for proline utilization A (PutA)

enzymes. PutAs are bifunctional enzymes that catalyze both reactions of

proline catabolism. Type A PutAs are the smallest members of the family,

possessing a minimal domain architecture consisting of N-terminal proline

dehydrogenase and C-terminal L-glutamate-c-semialdehyde dehydrogenase

modules. Type A PutAs form domain-swapped dimers, and in one case

(Bradyrhizobium japonicum PutA), two of the dimers assemble into a ring-

shaped tetramer. Whereas the dimer has a clear role in substrate channel-

ing, the functional significance of the tetramer is unknown. To address this

question, we performed structural studies of four-type A PutAs from two

clades of the PutA tree. The crystal structure of Bdellovibrio bacteriovorus

PutA covalently inactivated by N-propargylglycine revealed a fold and sub-

strate-channeling tunnel similar to other PutAs. Small-angle X-ray scatter-

ing (SAXS) and analytical ultracentrifugation indicated that Bdellovibrio

PutA is dimeric in solution, in contrast to the prediction from crystal pack-

ing of a stable tetrameric assembly. SAXS studies of two other type A

PutAs from separate clades also suggested that the dimer predominates in

solution. To assess whether the tetramer of B. japonicum PutA is necessary

for catalytic function, a hot spot disruption mutant that cleanly produces

dimeric protein was generated. The dimeric variant exhibited kinetic

parameters similar to the wild-type enzyme. These results implicate the

domain-swapped dimer as the core structural and functional unit of type A

PutAs.

Enzymes

Proline dehydrogenase (EC 1.5.5.2); L-glutamate-c-semialdehyde dehydrogenase (EC 1.2.1.88).

Databases

The atomic coordinates and structure factor amplitudes have been deposited in the Protein Data

Bank under accession number 5UR2. The SAXS data have been deposited in the SASBDB under

the following accession codes: SASDCP3 (BbPutA), SASDCQ3 (DvPutA 1.5 mg�mL�1),

SASDCX3 (DvPutA 3.0 mg�mL�1), SASDCY3 (DvPutA 4.5 mg�mL�1), SASDCR3 (LpPutA

3.0 mg�mL�1), SASDCV3 (LpPutA 5.0 mg�mL�1), SASDCW3 (LpPutA 8.0 mg�mL�1),

SASDCS3 (BjPutA 2.3 mg�mL�1), SASDCT3 (BjPutA 4.7 mg�mL�1), SASDCU3 (BjPutA

7.0 mg�mL�1), SASDCZ3 (R51E 2.3 mg�mL�1), SASDC24 (R51E 4.7 mg�mL�1), SASDC34

(R51E 7.0 mg�mL�1).

3029The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies

http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/5/5/2.html

http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/1/88.html

http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5UR2

Introduction

The proline catabolic pathway in bacteria and eukary-

otes consists of the enzymes proline dehydrogenase

(PRODH) and L-glutamate-c-semialdehyde aldehyde

dehydrogenase (GSALDH) (Fig. 1) [1]. PRODH cat-

alyzes the FAD-dependent oxidation of L-proline to

D1-pyrroline-5-carboxylate (P5C). Hydrolysis of P5C

generates the substrate for GSALDH, L-glutamate-c-semialdehyde. GSALDH is an aldehyde dehydrogenase

(ALDH) superfamily enzyme that catalyzes the

NAD+-dependent oxidation of L-glutamate-c-semial-

dehyde to L-glutamate. In total, the pathway produces

a 4-electron oxidation. The electrons abstracted from

proline flow into the electron transport chain, while

the carbon skeleton of L-proline ultimately enters the

citric acid cycle as a-ketoglutarate. Proline catabolic

enzymes have been implicated in many aspects of

health and disease, including tumor suppression [2],

hyperprolinemia metabolic disorders [3], schizophrenia

susceptibility [4–8], life-span extension [9], production

of fungal virulence factors [10], and virulence and sur-

vival of pathogenic bacteria [11–14].PRODH and GSALSH (aka P5CDH and

ALDH4A1) are combined in some bacteria into a sin-

gle protein known as proline utilization A (PutA)

[1,15]. The name PutA refers to early studies of proline

utilization in bacteria, which led to the discovery that

a single gene encodes both PRODH and GSALDH

[16,17]. The covalent linking of two enzymes into one

polypeptide chain suggests the possibility of substrate

channeling, which can improve kinetic efficiency, pro-

tect reactive intermediates, and prevent crosstalk

between competing pathways, such as proline catabo-

lism and biosynthesis [18]. Indeed, kinetic evidence in

support of substrate channeling has been found for

several PutAs [19–23].PutAs can be classified according to global sequence

similarity and domain architectures (Fig. 2). The PutA

phylogenetic tree has three prominent branches (1, 2,

and 3), and there are three types of PutA domain

architectures (A, B, and C). Type A PutAs have N-

terminal PRODH and C-terminal GSALDH modules

(Fig. 2A). Type B PutAs have an additional C-term-

inal domain, which was recently shown to have the

ALDH superfamily fold [24,25]. Type C PutAs have

an N-terminal ribbon-helix-helix DNA-binding domain

in addition to the C-terminal domain. These PutAs

function as transcriptional repressors of the genes

encoding PutA and the proline transporter PutP.

Types A, B, and C PutAs are found in branch 1

(Fig. 2B). Only type A PutAs are found in branch 2,

and only type B PutAs are observed in branch 3.

Thus, combining the phylogenetic tree and domain

analysis yields five classes of PutA: 1A, 1B, 1C, 2A,

and 3B.

The PutAs studied to date show surprising diversity

in oligomeric state and quaternary structure. Crystal

structures and oligomeric states in solution have been

determined for the class 1A PutA from Bradyrhizo-

bium japonicum (BjPutA) [20], the class 2A PutA from

Geobacter sulfurreducens (GsPutA) [23], the class 1B

PutA from Sinorhizobium meliloti (SmPutA) [24], and

the class 3B PutA from Corynebacterium freiburgense

(CfPutA) [25]. These structures showed that the

PRODH and GSALDH active sites are spatially sepa-

rated and connected by a tunnel. Mutagenesis studies

showed the tunnel is used for substrate channeling [26].

Although the arrangement of domains within the pro-

tomer is similar in all three structures, the oligomeric

states and quaternary structures differ. GsPutA forms a

domain-swapped dimer in solution. In BjPutA – anothertype A PutA – two of the dimers assemble into a ring-

shaped tetramer. The type B PutAs SmPutA and

CfPutA form monomer–dimer equilibria in solution;

however, the quaternary structure of the dimer is com-

pletely different from the type A domain-swapped dimer

[24,25]. Small-angle X-ray scattering (SAXS) showed

that the class 1C PutA from Escherichia coli forms yet a

third type of dimer, which is mediated by the DNA-

binding domain [27]. The functional relevance of these

various oligomeric states and quaternary structures has

not been systematically studied in detail.

The observation of different oligomeric states in

two-type A PutAs from different branches of the phy-

logenetic tree motivated us to study the self-association

of other type A PutAs to understand the relationship

between oligomeric state and catalytic function. In

particular, we sought to determine whether oligomeric

state distinguishes class 1A from class 2A and whether

Abbreviations

ALDH, aldehyde dehydrogenase; BbPutA, Bdellovibrio bacteriovorus proline utilization A; BCA, bicinchoninic acid assay; BjPutA,

Bradyrhizobium japonicum proline utilization A; CfPutA, Corynebacterium freiburgense proline utilization A; DvPutA, Desulfovibrio vulgaris

proline utilization A; GSALDH, L-glutamate-c-semialdehyde dehydrogenase; GsPutA, Geobacter sulfurreducens proline utilization A; LpPutA,

Legionella pneumophila proline utilization A; P5C, D1-pyrroline-5-carboxylate; PDB, Protein Data Bank; PRODH, proline dehydrogenase; PutA,

proline utilization A; SAXS, small-angle X-ray scattering; SEC, size exclusion chromatography; SmPutA, Sinorhizobiummeliloti proline

utilization A; TEVP, tobacco etch virus protease; THP, tris(3-hydroxypropyl)phosphine.

3030 The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies

Conservation of a core dimer in type A PutAs D. A. Korasick et al.

tetramerization of BjPutA is required for catalytic

activity.

Herein, we report a crystal structure of the class 2A

PutA from Bdellovibrio bacteriovorus (BbPutA), along

with SAXS data for BbPutA and three other type A

PutAs (from classes 1A and 2A). We also employed

analytical ultracentrifugation, size-exclusion chro-

matography, and enzyme kinetics for an extensive bio-

physical characterization of these enzymes. The data

suggest type A PutAs are predominantly dimeric in

solution, and global sequence identity is not a reliable

indicator of the oligomeric state. Further, we investi-

gate the necessity of higher order oligomeric states by

generating a dimeric hot spot mutant of the tetrameric

BjPutA. Overall, these results reveal a conserved dimer

as the essential oligomer required for catalysis and

channeling in type A PutAs. Our results also demon-

strate the challenges of predicting the solution oligo-

meric state from crystal packing.

Results

Crystal structure of covalently inactivated

BbPutA

The crystal structure of BbPutA inactivated by the

mechanism-based inactivator N-propargylglycine

(NPPG) was determined at 2.2 �A resolution (Table 1).

Similar to other PutAs, BbPutA has spatially sepa-

rated PRODH and GSALDH active sites (Fig. 3A).

The PRODH active site resides at the C termini of the

strands of a (ba)8 barrel. The GSALDH active site is

located in the crevice between the Rossmann dinu-

cleotide–binding domain and the GSALDH catalytic

domain. In addition to the catalytic domains, the fold

includes an N-terminal arm domain and an a-domain

in the PRODH half of the chain, as well as an

oligomerization flap at the C terminus. These ancillary

domains are also observed in other type A PutA struc-

tures. The root mean square deviation between

BbPutA and GsPutA is 1.1 �A, as expected for two

proteins with high sequence identity (47%, Table 2).

Fig. 1. The reactions catalyzed by PutA. PutAs consist of two modules that catalyze the oxidation of L-proline to L-glutamate. The first

module, proline dehydrogenase (PRODH), utilizes FAD to catalyze the conversion of L-proline to D1-pyrroline-5-carboxylate, which is subject

to nonenzymatic hydrolysis to L-glutamate-c-semialdehyde. The second module, L-glutamate-c-semialdehyde dehydrogenase (GSALDH),

performs the NAD+-dependent oxidation of L-glutamate-c-semialdehyde to the final product, L-glutamate.

Fig. 2. Classification of PutAs according to domain architecture

and global sequence identity. (A) The three domain architectures of

PutAs. RHH, ribbon-helix-helix; ALDHSF, aldehyde dehydrogenase

superfamily. The small N-terminal domain of type C PutAs is a

ribbon–helix–helix DNA-binding domain. (B) Phylogenetic tree

based on a global sequence alignment of PutAs. Architecture types

A, B, and C are indicated by black, blue, and red font, respectively.

The PutAs mentioned in the text are noted in large font. The

alignment was calculated with CLUSTAL OMEGA [75] and visualized

with DRAWTREE [76]. Abbreviation not listed in the text: EcPutA,

Escherichia coli proline utilization A.


D. A. Korasick et al. Conservation of a core dimer in type A PutAs

The deviations of BbPutA from BjPutA (class 1A) and

type B PutAs are also low (1.8–2.0 �A), despite sharing

only ~ 30% sequence identity with these proteins. This

result attests to the high structural conservation of the

PutA fold.

BbPutA has a substrate-channeling tunnel. The

FAD in the PRODH active site and the catalytic Cys

(Cys778) of the GSALDH site are separated by a lin-

ear distance of 45 �A and connected by a 65-�A curved

tunnel that varies in diameter from 1.5 �A near the

active sites to 4.5 �A in the middle section (Fig. 3A).

The dimensions of the tunnel are similar to those of

other PutAs. A coupled PRODH-GSALDH enzyme

activity assay, which tests both active sites and pro-

vides preliminary evidence of substrate channeling [26],

Table 1. Data collection and refinement statisticsa.

Space group P21221

Beamline APS 24-ID-E

Unit cell parameters (�A) a = 144.3, b = 158.6, c = 221.1

Wavelength (�A) 0.97920

Resolution (�A) 128.9–2.23 (2.35–2.23)

Observations 930 561

Unique reflections 241 211

Rmerge (I) 0.095 (0.493)

Rmeas (I) 0.124 (0.644)

Rpim (I) 0.061 (0.320)

Mean I/r 10.0 (2.4)

Completeness (%) 97.9 (98.3)

Multiplicity 3.9 (3.9)

No. of protein residues 3829

No. of atoms

Protein 29 525

FAD 212

Modified Lys 48

Water 1048

Rcryst 0.183 (0.230)

Rfreeb 0.228 (0.291)

rmsd bond lengths (�A) 0.008

rmsd bond angles (°) 0.872

Ramachandran plotc

Favored (%) 97.95

Outliers (%) 0.00

Clashscore (PR)c 1.73 (100)

MolProbity score (PR)c 0.94 (100)

Average B (�A2)

Protein 31.2

FAD 31.4

Modified Lys 43.0

Water 30.2

Coordinate error (�A)d 0.27

PDB code 5UR2

a Values for the outer resolution shell of data are given in parenthe-

ses. b 5% test set. c From MOLPROBITY. The percentile ranks (PR) for

Clashscore and MolProbity score are given in parentheses. d Maxi-

mum likelihood-based coordinate error estimate from PHENIX.

A

B

C E

D

Fig. 3. Structure of BbPutA. (A) A protomer of BbPutA with the

domains colored according to the domain diagram. The pink surface

represents the substrate-channeling tunnel, which connects the two

active sites. The FAD is shown in yellow sticks. Catalytic Cys778 is

drawn in spheres. (B) The domain-swapped dimer of BbPutA. The

two protomers have different colors. The molecular two-fold axis is

vertical. (C) Close-up view of a portion of the dimer interface where

the oligomerization flap of one protomer covers the substrate-

channeling tunnel of the opposite protomer. (D) Surface

representation of the dimer interface shown in panel C, highlighting

how dimerization seals the substrate-channeling tunnel from the

bulk medium. (E) Results of a PRODH-GSALDH-coupled assay for

BbPutA. The reaction mixture contained BbPutA (0.2 lM), proline

(40 mM), menadione bisulfite (0.1 mM), and NAD+ (0.2 mM) in a

buffer containing 50 mM potassium phosphate, 25 mM NaCl, and

10 mM MgCl2 at pH 7.5. Production of NADH was monitored at

340 nm. The data points represent the average of assays performed

in triplicate. [Colour figure can be viewed at wileyonlinelibrary.com]



http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5UR2

confirmed that both actives sites are functional

(Fig. 3E). In addition, no lag phase is apparent in the

time-dependence of NADH production, consistent

with the presence of a functional substrate-channeling

tunnel.

The structure of BbPutA was determined from a

crystal of the enzyme that had been inactivated with

NPPG (Fig. 4A, 1). Previous studies of NPPG-inacti-

vated PRODHs and PutAs show that inactivation is

due to a covalent link between the FAD N5 atom and

a conserved active site Lys (Fig. 4A, 2). Electron den-

sity maps for BbPutA show evidence of this inactiva-

tion mechanism. An electron density feature connects

the e-amino group of Lys196 with the N5 atom of the

FAD (Fig. 4B). The strength of this feature varied

among the four chains in the asymmetric unit, with

the strongest density observed in chain D (Fig. 4). The

electron density feature could be modeled satisfactorily

as a 3-carbon link between Lys196 and the N5 of the

FAD consistent with the presence of a covalent NPPG

modification in the active site.

The FAD exhibits the structural hallmarks of reduc-

tion. Previous studies revealed that NPPG locks the

flavin into a conformation that resembles the 2-elec-

tron reduced state [28]. These features are seen in the

BbPutA structure. In particular, the isoalloxazine moi-

ety exhibits a butterfly bend of 25° (si face convex)

(Fig. 4B). For reference, the bend angles of other

NPPG-inactivated PutAs and PRODHs are 25°–35°[23,28,29]. Furthermore, the ribityl chain conformation

of NPPG-inactivated BbPutA is indicative of reduced

PutA/PRODH. The 20-OH and 30-OH groups of the

inactivated FAD are rotated toward the pyrimidine

side of the isoalloxazine, while the 40-OH is beneath

the dimethylbenzene ring (Fig. 4C). This conformation

is also observed in other NPPG-inactivated or dithion-

ite-reduced PutAs [23,28] and PRODHs [29,30].

Solution oligomeric state analysis of BbPutA by

SAXS and analytical ultracentrifugation

The oligomeric state and quaternary structure of

BbPutA in solution were determined using SAXS and

analytical ultracentrifugation (Fig. 5, Table 3). Guinier

analysis of experimental SAXS data yields a radius of

gyration (Rg) of 45 �A (Fig. 5A). For reference, the 2-

body assembly in the crystallographic asymmetric unit

also has Rg of 45 �A (Fig. 3B). The real space Rg from

calculations of the distance distribution function is

45.9–46.7 �A for assumed maximum particle dimension

(Dmax) of 140–150 �A (Fig. 5B, Table 3). Thus, the

reciprocal space and real space radii of gyration are in

good agreement. The oligomeric state was estimated

using the volume of correlation method [31]. This

analysis yields molecular mass (Mr) of 190 kDa, which

is within 13% of the theoretical Mr of a dimer

(218 kDa, Table 3). Similarly, Mr estimated from the

SAXS MoW2 server [32] is 226 kDa, which is within

4% of the dimer (Table 3). Altogether, the SAXS data

are consistent with BbPutA forming a dimer under the

solution conditions and protein concentration

(1.8 mg�mL�1) used for SAXS.

The BbPutA crystal lattice was inspected using PDBe-

PISA [33] to identify plausible oligomers. This analysis

revealed two stable assemblies, including the classic

type A PutA domain-swapped dimer (Rg = 45 �A,

Figs 3B and 5C) and a ring-shaped tetramer

(Rg = 54 �A, Fig. 5D). The predicted tetramer is gener-

ated by a crystallographic 2-fold rotation applied to

the domain-swapped dimer. Thus, the tetramer is a

Table 2. Pairwise amino acid sequence identities of type A PutAs.

LpPutA BjPutA BbPutA GsPutA DvPutA

LpPutA 100 50 31 31 30

BjPutA 100 32 31 29

BbPutA 100 47 48

GsPutA 100 67

Fig. 4. Covalent modification of the FAD. (A) Structures of (1)

NPPG and (2) the covalently modified FAD resulting from

inactivation by NPPG. (B) Electron density evidence of inactivation

of BbPutA by NPPG. This view of the N5 edge of the isoalloxazine

shows the 25° butterfly bend induced by inactivation. The mesh

represents a simulated annealing Fo-Fc omit map contoured at 2.5

r. (C) Electron density for the modified FAD of BbPutA. The mesh

represents a simulated annealing Fo-Fc omit map contoured at

2.5 r.



dimer-of-dimers and has 222 point group symmetry.

We note this tetramer resembles the one formed by

BjPutA in solution; however, the BbPutA tetramer is

slightly larger (Rg of 54 �A vs. 51 �A for BjPutA). In

addition, PDBePISA analysis returned an octamer

consisting of two of the 54 �A tetramers, which was

classified in the uncertain region of complex formation

criteria (Rg = 68 �A). The experimental Rg of 45–47 �A

suggests that the dimer is the predominant species in

solution. FoXS [34,35] was used to assess the agree-

ments of the dimer and tetramer models to the

experimental curve. The scattering curve calculated

from the dimer has a goodness-of-fit parameter (v) of1.3, whereas the curve calculated from the crystallo-

graphic tetramer has a much larger v of 18 (Fig. 5A).

Monomer–dimer and dimer–tetramer equilibria were

explored using MultiFoXS [35]. MultiFoXS returned

neither monomer–dimer nor dimer–tetramer 2-body

fits, indicating that 100% dimer provides the best

interpretation of the SAXS data. Finally, shape recon-

struction with DAMMIF [36] produced a shape consistent

with the crystallographic dimer (Fig. 5C). In summary,

Fig. 5. SAXS and analytical ultracentrifugation of BbPutA. (A) Experimental SAXS curve measured at 1.8 mg�mL�1 (open circles). The inset

shows a Guinier plot. Theoretical curves calculated from the BbPutA dimer (C) and tetramer (D) are shown in solid red (FoXS v = 1.3) and

red dashes (FoXS v = 18.1), respectively. (B) Experimental SAXS distance distribution function. (C) The crystallographic dimer of BbPutA.

The surface represents the shape reconstruction from DAMMIF. The correlation coefficient between the crystal structure and the shape

reconstruction volumetric map is 0.85. (D) The BbPutA tetramer predicted by PDBePISA from analysis of crystal packing. (E) Sedimentation

velocity analysis for BbPutA at 4 mg�mL�1 (~ 37 lM).



SAXS suggests that BbPutA is primarily dimeric in

solution, and furthermore, the 2-body assembly in the

asymmetric unit is the predominant species formed in

solution.

Analytical ultracentrifugation was employed to

determine whether BbPutA self-associates into higher

oligomeric states at a higher concentration than was

used in SAXS. A sedimentation velocity experiment

performed at 4 mg�mL�1 (~ 37 lM) revealed a distri-

bution of sedimentation coefficients with a major peak

at approximately 6.4 S. This apparent sedimentation

coefficient corresponds to Mr of approximately

225 kDa (Fig. 5E). The expected Mr of a BbPutA

dimer is 218.6 kDa.

Analytical ultracentrifugation was also used to test

whether inactivation with NPPG induces tetrameriza-

tion. This experiment was motivated by the observa-

tion of an apparent tetramer in the crystal structure of

NPPG-inactivated BbPutA (Fig. 5D). Sedimentation

velocity analysis of NPPG-inactivated BbPutA

revealed one major peak corresponding to Mr of

225 kDa (Fig. 5E), showing that inactivation does not

promote tetramerization in solution.

Finally, sedimentation velocity was performed on

BbPutA in the presence of the active site ligands L-te-

trahydro-2-furoic acid (THFA, 10 mM) and NAD+

(1 mM). THFA is a competitive inhibitor (competitive

with proline) of PutAs and is known to occupy the

proline-binding site of PutAs and monofunctional

PRODHs [23,24,30,37,38]. NAD+ is the cofactor of

the GSALDH reaction. This experiment was moti-

vated by our recent observation that THFA and

NAD+ induce monomers of the class 3B CfPutA to

form dimers [25]. The c(s) distribution obtained for

BbPutA under this condition exhibited a single c(s)

peak at 6.4 S, corresponding to Mr of 225 kDa

Table 3. Structural and molecular mass (Mr) parameters from SAXS for BbPutA, DvPutA and LpPutA.

BbPutA 1.8 mg�mL�1

Rg from Guinier (�A) 44.9 � 0.4

I(0) from Guinier (�A�1) 1130 � 7

Dmax (�A) 140–150

Rg from P(r) (�A)a 45.9–46.7

I(0) from P(r) (�A�1)a 1117–1132

Porod volume (�A3)a 288 000–289 000

Volume of correlation, Vc (�A2)b 1034

Mr from Vc (kDa)b 190

Mr from MoW2 (kDa)c 226

Monomeric Mr from sequence 109

DvPutA 1.5 mg�mL�1 3.0 mg�mL�1 4.5 mg�mL�1

Rg from Guinier (�A) 43.6 � 0.4 43.6 � 0.3 43.9 � 0.3

I(0) from Guinier (�A�1) 681 � 4 1600 � 8 2319 � 11

Dmax (�A) 150–160 150–160 150–160

Rg from P(r) (�A)a 46.1–47.0 45.3–45.7 45.7–46.5

I(0) from P(r) (�A�1)a 699–703 1623–1627 2350–2364

Porod volume (�A3)a 292 000–294 000 294 000–295 000 294 000–295 000

Volume of correlation, Vc (�A2)b 994 1020 1010

Mr from Vc (kDa)b 180 190 180

Mr from MoW2 (kDa)c 213 224 222

Monomeric Mr from sequence 112 112 112

LpPutA 3 mg�mL�1 5 mg�mL�1 8 mg�mL�1

Rg from Guinier (�A) 45.5 � 0.2 45.9 � 0.1 46.3 � 0.1

I(0) from Guinier (�A�1) 471 � 1 1010 � 1 1475 � 0.2

Dmax (�A) 150–160 150–166 150–170

Rg from P(r) (�A)a 46.5–46.9 46.6–46.9 46.7–47.2

I(0) from P(r) (�A�1)a 473–475 1014–1018 1470–1481

Porod volume (�A3)a 290 000–291 000 297 000–298 000 294 000–296 000





a From calculations of P(r) using PRIMUS [64] with the indicated range of Dmax.b Calculated with Scatter 3.0 [70]. c Calculated from the

method of Fischer et al. [32].



(Fig. 5E), showing that the binding of THFA and

NAD+ does not induce higher order oligomerization.

Taken together, the SAXS and analytical ultracentrifu-

gation results are consistent with BbPutA being pri-

marily dimeric under solution conditions that are

relevant to catalytic function.

SAXS analysis of two other type A PutAs

To further explore the oligomerization of type A

PutAs, SAXS was performed on the class 2A PutA

from Desulfovibrio vulgaris (DvPutA) and the class 1A

PutA from Legionella pneumophila (LpPutA). DvPutA

is 48% identical in amino acid sequence to BbPutA

and 67% identical to GsPutA (Table 2). Such high

sequence homology is expected from PutAs of the

same clade (branch 2, Fig. 2B). In contrast, LpPutA, a

branch 1 PutA, is only 30% identical to the class 2A

PutAs, but 50% identical to the class 1A BjPutA

(Table 2). Analysis of DvPutA and LpPutA provides

two more data points on the relationship between

sequence and oligomeric state for type A PutAs.

SAXS curves measured at three different concentra-

tions of DvPutA and LpPutA are shown in Fig. 6.

SAXS-derived parameters are listed in Table 3. The

average Rg from Guinier analysis is 44 �A for DvPutA

and 46 �A for LpPutA. These values agree more closely

with the Rg of 45 �A of dimeric BbPutA than with the

Rg expected for a tetrameric type A PutA (51–54 �A).

The distance distribution functions of DvPutA and

LpPutA resemble that of BbPutA. For all three pro-

teins, the distribution has a major peak near the real-

space vector length (r) of 40 �A followed by a shoulder

peak at r = 80–100 �A (Figs 5B and 6B,D), suggesting

the three proteins share a common quaternary struc-

ture. Further, the SAXS-derived molecular masses of

DvPutA and LpPutA based on the volume of correla-

tion are within 20% of the theoretical dimer masses

for each protein (Table 3). Moreover, the masses from

the SAXS MoW2 server are within 4% of the theoreti-

cal dimer mass (Table 3). Note also the Porod volumes

for DvPutA and LpPutA (290 000–298 000 �A3) are

similar to that of BbPutA (289 000 �A3) and almost

two times smaller than expected for tetrameric PutA

(~ 560 000 �A3). The theoretical SAXS curves calcu-

lated from domain-swapped dimeric homology models

show good agreement with the experimental profiles in

the region of q = 0–0.1 �A�1 (Fig. 6). We note that this

region is critical for distinguishing between dimeric

and tetrameric PutAs (see Fig. 5A and references

[20,23]). Monomer–dimer and dimer–tetramer equilib-

ria were explored using MultiFoXS [35]. As with

BbPutA, MultiFoXS returned neither monomer–dimer

nor dimer–tetramer 2-body fits, indicating that 100%

dimer provides the best interpretation of the SAXS

data for DvPutA and LpPutA. Finally, shape recon-

structions generated shapes that are consistent with

the dimeric models and do not resemble a ring-shaped

particle (Fig 6A,C). Altogether, the SAXS results sug-

gest that DvPutA and LpPutA are dimeric in solution

and form the classic type A PutA domain-swapped

dimer.

Identification of the core functional oligomer of

BjPutA

All type A PutAs studied thus far appear to be dimeric

in solution except for BjPutA, which is tetrameric

(Fig. 7A) [20]. To better understand whether the tetra-

meric assembly is necessary for BjPutA function, we

attempted to generate a hot spot mutant to disrupt

tetramerization. Structural analysis of BjPutA revealed

Arg51 as potentially important for stabilizing the tetra-

mer. Arg51 is located in the dimer–dimer interface on

a flexible loop that connects the N-terminal arm to the

a-domain (Fig. 7B). Although Arg51 has weak elec-

tron density and appears to make no strong interdo-

main hydrogen bonds or ion pairs, its location in the

center of the dimer–dimer interface nevertheless sug-

gested that it could be important for tetramer forma-

tion. Analysis with PDBePISA indicates that Arg51

contributes 76 �A2 of surface area to the tetramer inter-

face, which makes it the second-largest contributor,

behind only Tyr474 (99 �A2). Interestingly, Arg does

not appear at this position in the sequences of the

dimeric type A PutAs studied so far; rather, it is

replaced with Glu in LpPutA, Gln in BbPutA, and

Gly in GsPutA and DvPutA. Therefore, we generated

a mutant, R51E, to reflect the charge reversal seen in

LpPutA. We note the introduction of Glu51 in BjPutA

results in three consecutive acidic residues: Glu51-

Asp52-Asp53.

To understand any effects of this mutation on the

quaternary structure of BjPutA, we first purified wild-

type BjPutA and subjected it to analysis by sedimenta-

tion velocity. Initial sedimentation velocity studies of

wild-type BjPutA revealed a major peak near apparent

sedimentation coefficient of 10.8 S (Fig. 8A), which

corresponds to Mr of 429 kDa (Fig. 8B). The pre-

dicted Mr of the BjPutA tetramer is also 429 kDa.

Wild-type BjPutA was also studied with SAXS. The

SAXS curves for wild-type BjPutA show pronounced

trough and peak features in the region of q = 0.05–0.1 �A�1 (Fig. 8C). We have shown previously that

these features are diagnostic of the ring-shaped tetra-

meric form of type A PutA [20,23]. Note these



diagnostic features are absent in the SAXS curves for

all the other PutAs studied here. Also, the distance dis-

tribution function of wild-type BjPutA is very different

from those of the other proteins in this study

(Fig. 8D). The distribution function of wild-type

BjPutA indicates a most-probable real-space vector

length of r ~ 80 �A (Fig. 8D), compared to approxi-

mately 40 �A for the other PutAs (Figs 5B and 6B,D).

The SAXS Rg of 51–53 �A for wild-type BjPutA

(Table 4) is in good agreement with the Rg of 51 �A

calculated from the BjPutA tetramer. The molecular

masses derived from SAXS are within 0.5–11% of the

expected mass of a tetramer (Table 4). The SAXS

curve calculated from the BjPutA tetramer shows good

agreement with the experimental curves (v = 4.3–9.1,Fig. 8C). The fits could be improved somewhat (to

v = 1.6–4.4) by using a tetramer : dimer ensemble in

MultiFoXS (Fig. 8C). The optimal ratio of

Fig. 6. SAXS analysis of DvPutA and LpPutA. (A) Experimental SAXS curves for DvPutA (open circles) at three concentrations: 1.5, 3.0, and

4.5 mg�mL�1. The inset shows Guinier plots. The red curve was calculated from a homology model of the DvPutA domain-swapped dimer

(shown in the inset). The v values obtained from FoXS fits of the theoretical scattering curve of the homology model to the experimental

scattering data are as follows: v = 1.3 (1.5 mg�mL�1), v = 1.7 (3.0 mg�mL�1), and v = 2.3 (4.5 mg�mL�1). A homology model of the DvPutA

dimer is shown inside the DAMMIF shape reconstruction. The correlation coefficient between the homology model and the shape

reconstruction volumetric map is 0.79. (B) Experimental SAXS distance distribution functions for DvPutA. (C) SAXS curves for LpPutA (3, 5,

8 mg�mL�1). The inset shows Guinier plots. The red curve was calculated from a homology model of the LpPutA domain-swapped dimer

(shown in the inset). The v values obtained from FoXS fits of the theoretical scattering curve of the homology model to the experimental

scattering data are as follows: v = 3.3 (3.0 mg�mL�1), v = 4.9 (5.0 mg�mL�1), and v = 8.7 (8.0 mg�mL�1). A homology model of the LpPutA

dimer is shown inside the DAMMIF shape reconstruction. The correlation coefficient between the homology model and the shape

reconstruction volumetric map is 0.78. (D) Experimental SAXS distance distribution functions for LpPutA.



tetramer : dimer ranged from 74%:26% for the lowest

concentration sample to 81%:19% for the highest con-

centration sample. Thus, the fitting calculations are

consistent with BjPutA being predominantly tetrameric

in solution. Finally, shape reconstruction calculations

using the SAXS data for the highest concentration

sample returned a ring-shaped particle consistent with

the crystallographic tetramer of BjPutA (Fig. 8C). In

summary, the SAXS data show that wild-type BjPutA

exists in solution primarily as a ring-shaped tetramer,

in agreement with our previously crystallographic,

SAXS, and centrifugation studies [20,23].

The mutation of Arg51 to Glu profoundly changes

the oligomeric structure of BjPutA. To determine the

effects of the R51E mutation on the oligomeric state

of BjPutA, we subjected the BjPutA R51E mutant

variant to the same analysis as wild-type BjPutA. Sedi-

mentation velocity experiments on BjPutA R51E

revealed a major peak at apparent sedimentation coef-

ficient of 6.5 S, which corresponds to Mr of 216 kDa

(Fig 8A,B). The theoretical Mr for the BjPutA dimer

is 214.7 kDa, which is within 0.6% of the result from

sedimentation velocity, suggesting BjPutA R51E is

dimeric in solution. SAXS analysis of BjPutA R51E at

three protein concentrations revealed experimental

curves statistically very similar (v = 0.9–2.1) to the

theoretical curves generated by FoXS using the

crystallographic domain-swapped dimer (Fig. 8E).

Monomer–dimer and dimer–tetramer equilibria were

explored using MultiFoXS [35]. MultiFoXS returned

neither monomer–dimer nor dimer–tetramer 2-body

fits, indicating that 100% dimer provides the best

interpretation of the SAXS data for R51E. Guinier

analysis returned Rg of 45 �A, consistent with the Rg of

dimeric type A PutAs (Table 4). The real-space Rg of

45–46 �A agrees well with the Guinier Rg. The P(r) dis-

tribution for R51E (Fig. 8F) is distinctly different

from that of wild-type BjPutA (Fig. 8D). Further, fit-

ting a representative experimental R51E SAXS curve

to the BjPutA crystallographic dimer yields a greatly

improved statistical fit (v = 1.3) compared with fitting

the experimental data to the BjPutA tetramer (v = 51;

Fig. 9A). Moreover, the experimental P(r) distribu-

tions of wild-type BjPutA and R51E are qualitatively

very different (Fig. 9B). In fact, the area under the dis-

tance distribution function of R51E is approximately

Fig. 7. Structural context of Arg51 of BjPutA. (A) The BjPutA

tetramer with the four protomers in different colors. The domain-

swapped dimers are colored red-cyan and gold-blue. Arg51 is

shown in spheres in the right-hand image. The boxed region is

expanded in panel B. (B) Close-up view of the dimer–dimer

interface. The dashed curves represent disordered residues 52–53.

Fig. 8. Solution oligomeric state analysis of BjPutA and BjPutA R51E. (A) The distribution of apparent sedimentation coefficients from

sedimentation velocity observed for BjPutA (28 lM, black) or BjPutA R51E (28 lM, red). (B) The distribution of molecular masses from

sedimentation velocity observed for BjPutA (28 lM, black) or BjPutA R51E (28 lM, red). (C) SAXS curves measured at three wild-type BjPutA

protein concentrations (2.3, 4.7, 7.0 mg�mL�1). The inset shows Guinier plots. The theoretical SAXS curve calculated from the crystallographic

tetramer (inset) is shown in cyan [v values of 4.3 (2.3 mg�mL�1), 7.3 (4.7 mg�mL�1), and 9.1 (7.0 mg�mL�1)]. The theoretical curves obtained

from MultiFoXS assuming a mixture of the crystallographic tetramer and dimer are shown in red [v values of 1.6 (2.3 mg�mL�1), 3.3

(4.7 mg�mL�1), and 4.4 (7.0 mg�mL�1)]. The optimal tetramer : dimer compositions from MultiFoXS are 74%:26% (2.3 mg�mL�1), 77%:23%

(4.7 mg�mL�1), and 81%:19% (7.0 mg�mL�1). The crystallographic tetramer of BjPutA is shown inside the DAMMIF shape reconstruction. The

correlation coefficient between the tetramer and the shape reconstruction volumetric map is 0.78. (D) Experimental SAXS distance distribution

functions for wild-type BjPutA. (E) SAXS curves measured at three BjPutA R51E protein concentrations. The inset shows Guinier plots. The

theoretical SAXS curve calculated from the BjPutA domain-swapped dimer (inset) is shown in red. The v values obtained from FoXS fits of the

theoretical scattering curve of the crystallographic dimer to the experimental scattering data are as follows: v = 0.85 (2.3 mg�mL�1), v = 1.3

(4.7 mg�mL�1), and v = 2.5 (7.0 mg�mL�1). The crystallographic dimer of BjPutA is shown inside the DAMMIF shape reconstruction. The

correlation coefficient between the tetramer and the shape reconstruction volumetric map is 0.83. (F) Experimental SAXS distance distribution

functions for BjPutA R51E.



54% of the area under the distance distribution func-

tion of wild-type BjPutA at the same protein concen-

tration, consistent with R51E being half the size of

wild-type BjPutA (Fig. 9B). Overall, these results sug-

gest that the R51E mutation cleanly disrupts tetramer-

ization, resulting in consistently dimeric BjPutA.



To determine whether the R51E mutation has an

effect on the catalytic activity, we performed the cou-

pled PRODH-GSALDH activity assay, which reports

on both catalytic activities and provides information

on substrate channeling. Previous results have shown

that at high concentrations of proline, kinetic data for

the coupled PRODH-GSALDH assay fit to a substrate

inhibition model [26]. Substrate inhibition was also

observed here for both wild-type BjPutA and R51E

(Fig. 10A,B). Using proline as the variable substrate,

wild-type BjPutA displayed a Km of 4 mM, kcat of

0.31 s�1, and inhibition constant for proline (Ki) of

400 mM (Fig. 10A, Table 5). Similarly, the kinetic

parameters for R51E are Km = 10 mM, kcat = 0.4 s�1,

and Ki of 220 mM (Fig. 10B, Table 5). The catalytic

efficiencies (kcat/Km) of wild-type BjPutA and R51E

are within a factor of 2 (Table 5). These results suggest

that tetramerization is not essential for the in vitro cat-

alytic activity of BjPutA.

We considered the hypothesis that dimeric R51E

assembles into the tetramer under the conditions of

the activity assay, i.e., that reduction of the FAD or

substrate binding enhances the association of two

dimers into the tetramer. To test this idea, we first per-

formed sedimentation velocity experiments after inacti-

vation by NPPG. NPPG locks the FAD into the

reduced state, thus mimicking a major effect of proline

oxidation on the enzyme. NPPG-inactivated R51E

displayed an apparent sedimentation coefficient of 5.9

S (Fig. 10C), which is close to that of untreated R51E

(6.5 S) and far from that of wild-type BjPutA (10.8 S).

We next performed sedimentation velocity of R51E in

the presence of the active site ligands THFA and

NAD+, which bind in the PRODH and GSALDH

active sites, respectively. We note that the binding of

these ligands to a monomeric type B PutA (CfPutA)

induces dimerization [25]. R51E in the presence of

THFA and NAD+ exhibits an apparent sedimentation

coefficient (6.2 S) consistent with a dimer (Fig. 10C).

Thus, in contrast to monomeric type B PutA, the

binding of active site ligands does not induce higher

order assembly of R51E. Finally, we conducted the

coupled activity assay for R51E as a function the

enzyme concentration. The observed rate is linearly

proportional to R51E concentration, as expected for

an enzyme that does not require assembly into a

higher order oligomer for activity (Fig. 10D). Taken

together, our results suggest that R51E forms a cat-

alytically competent dimer.

Discussion

Almost 50% of proteins are homooligomers, implying

that self-association underlies function [39,40]. There

are many possible reasons for oligomerization. For

example, substrate and cofactor binding sites of

Table 4. Structural and molecular mass (Mr) parameters from SAXS for BjPutA and BjPutA R51E.

BjPutA 2.3 mg�mL�1 4.7 mg�mL�1 7.0 mg�mL�1

Rg from Guinier (�A) 53 � 2 52 � 1 51.9 � 0.7

I(0) from Guinier (�A�1) 183 � 8 409 � 7 607 � 7

Dmax (�A) 140–146 140–146 136–140

Rg from P(r) (�A)a 51.4–51.6 51.6–51.7 51.4–51.5

I(0) from P(r) (�A�1)a 177–180 406–412 612–613

Porod volume (�A3)a 541 000–549 000 553 000–561 000 560 000





BjPutA R51E 2.3 mg�mL�1 4.7 mg�mL�1 7.0 mg�mL�1

Rg from Guinier (�A) 44.9 � 0.7 45.0 � 0.6 44.9 � 0.4

I(0) from Guinier (�A�1) 118.9 � 0.4 232 � 3 356 � 3

Dmax (�A) 140–150 136–145 140–150

Rg from P(r) (�A)a 45.3–45.7 44.9–45.0 45.8–46.0

I(0) from P(r) (�A�1)a 116–117 224–227 356–358

Porod volume (�A3)a 280 000–281 000 278 000–283 000 288 000–289 000





a From calculations of P(r) using PRIMUS [64] with the indicated range of Dmax.b Calculated with SCATTER 3.0 [70]. c Calculated from the

method of Fischer et al. [32].



enzymes can occur in oligomer interfaces [41,42]. Even

when the active site is not in an interface, oligomeriza-

tion may still be essential for catalytic activity, as in

some ALDHs [43,44]. Many integral membrane trans-

porters function as oligomers [45]. Quaternary structure

is also important in allosteric proteins [46], cooperative

enzymes [47], and morpheein enzymes [48]. Oligomeriza-

tion can also contribute to protein stability [41].

Here, we investigated the functional significance of

oligomerization in type A PutAs. The first type A

PutA studied, BjPutA, was found to be a tetramer

consisting of a pair of domain-swapped dimers that

form a ring [20]. Herein, we showed that several other

type A PutAs are dimeric, implying the BjPutA tetra-

mer may be an exception.

The domain-swapped dimer observed in all type A

PutA crystal structures, whether as a stand-alone

dimer or half of a tetramer, has obvious functional rel-

evance. The oligomerization flap of one protomer seals

the substrate-channeling tunnel of the other protomer,

as shown for BbPutA (Fig. 3C,D). Without this inter-

molecular lid, the intermediate would have a higher

probability of diffusing into the bulk medium. Thus,

domain-swapped dimerization in type A PutAs enables

the substrate-channeling step of the catalytic mecha-

nism. We note that in type B PutAs, the intermolecu-

lar lid is replaced by an intramolecular (tertiary

structural) interaction involving the C-terminal ALDH

superfamily domain [21,24,25].

The functional significance of the BjPutA tetramer is

not obvious, prompting the work reported here. The

dimer–dimer interface is formed by the N-terminal

arm and a-domain, two regions of the protein that

lack catalytic residues and ligand-binding sites. The

interface is far from the active sites and substrate-

channeling tunnel. For example, the hot spot residue

Arg51 is 30 �A from the nearest flavin N5 and 50 �A

from the catalytic Cys of the GSALDH site. Consis-

tent with these observations, the dimeric hot spot vari-

ant R51E exhibits wild-type catalytic behavior and

displays an apparent sedimentation coefficient consis-

tent with the formation of a dimer in solution. The

sedimentation data for R51E contain no evidence for

tetramer formation. Further, it was observed that nei-

ther flavin reduction nor incubation with active site

ligands induces tetramer formation by R51E or

BbPutA (Figs 5E and 10C). Therefore, it is concluded

that tetramerization is not essential for catalytic

function, suggesting that the domain-swapped dimer is

the core oligomeric structure of type A PutAs. We

note that this conclusion is rendered from in vitro bio-

chemical and biophysical analysis of PutAs and that,

under cellular conditions, molecular crowding or other

favorable conditions may promote higher-order

oligomerization.

Our results for BjPutA are consistent with the hot

spot theory of protein–protein interaction [49,50]. Hot

spots refer to the region of a protein–protein interface

that contains a few critical residues that account for

most of the association energy. Hot spots have a dis-

tinctive amino acid composition – often Trp, Arg, or

Tyr [50]. Consistent with the hot spot theory, we could

abrogate tetramerization of BjPutA with the single

mutation of Arg51 to Glu. It is interesting that this

Fig. 9. Comparison of the in-solution properties of BjPutA and

BjPutA R51E. (A) A representative experimental SAXS curve of

BjPutA R51E (4.7 mg�mL�1) is shown in open circles. Overlaid are

the theoretical SAXS curves calculated from the BjPutA domain-

swapped dimer (solid red) and the BjPutA tetramer (dashed red).

The v values obtained from FoXS fits of the theoretical scattering

curve of the crystallographic dimer and crystallographic tetramer to

the experimental scattering data are 1.3 and 51.9, respectively. (B)

Experimental distance distributions for wild-type BjPutA (black) and

R51E (red).



particular Arg residue makes no specific electrostatic

interactions in the native tetramer (Fig. 7). Further-

more, Arg51 is located on a disordered loop. Thus,

visual inspection of the crystal structure might not

have revealed Arg51 as a hot spot residue. However,

analysis of the interface with PDBePISA indicated that

Arg51 contributes significantly to the dimer–dimer

interface surface area, despite the lack of the tradi-

tional electrostatic interactions one typically associates

with Arg. This result points to buried surface area as a

key metric for identifying hot spot residues.

In addition, our results indicate that the prediction

of quaternary structure and oligomeric state from crys-

tal packing remains challenging in some cases. The

Fig. 10. Kinetic data for wild-type BjPutA and R51E, and the effects of active site ligands on the oligomeric state of R51E. (A) Dependence

of the coupled PRODH-GSALDH reaction rate on proline concentration for wild-type BjPutA (black points). Kinetic data were fit to a

substrate inhibition model (red curve). (B) Dependence of the coupled PRODH-GSALDH reaction rate on proline concentration for R51E

(black points). Kinetic data were fit to a substrate inhibition model (red curve). The assays used fixed concentrations of proline (40 mM),

CoQ1 (0.1 mM), and NAD+ (0.2 mM). (C) The distribution of sedimentation coefficients observed from sedimentation velocity experiments of

R51E performed in the absence of ligands (solid black), after inactivation with NPPG (red), or in the presence of the active site ligands THFA

(10 mM) and NAD+ (1 mM) (blue). The distribution of sedimentation coefficients observed for wild-type BjPutA (dashed black) in the absence

of ligands is provided for reference. The enzyme concentration was 28 lM in all experiments. (D) Dependence of the coupled PRODH-

GSALDH reaction rate on protein concentration for R51E (black points). Data were fit to a linear regression model (red line).

Table 5. Kinetic Parameters for BjPutA and BjPutA R51E.

Km

(mM)

kcat

(s�1)

kcat/Km

(M�1�s�1) Ki (mM)

BjPutA 4 � 1 0.31 � 0.03 70 � 20 400 � 100

BjPutA R51E 10 � 2 0.40 � 0.04 40 � 10 220 � 50



PISA algorithm, which is based on physical–chemical

models of protein interactions and chemical thermody-

namics, is perhaps the gold standard for predicting oli-

gomeric state from crystal packing and is used by the

PDB to identify the most likely biological assembly

[33]. PISA analysis suggested that BbPutA forms a

stable tetramer in solution (Fig. 5D). However, our

SAXS and sedimentation data showed no evidence of

a tetramer at concentrations in the range of 1.8–4 mg�mL�1 (16–37 lM). Although it is possible that

the tetramer could form at even higher concentrations,

we have observed solubility problems with BbPutA at

concentrations above 10 mg�mL�1. It is possible that

the high protein concentration achieved as the crystal-

lization drop equilibrates promotes formation of the

higher order assembly seen in the crystal. Such high

concentrations are not possible in solution for

BbPutA, so the tetramer may not be observable with

standard solution biophysical techniques. Finally, it

remains possible that the tetramer could form in the

cell, where molecular crowding may enhance associa-

tion of dimers. We have also encountered this phe-

nomenon – where the crystal and solution data

conflict – with monofunctional GSALDHs [51]. In that

case, the wild-type enzyme is hexameric both in solu-

tion and in the crystal. However, mutation of a hex-

amerization hot spot residue produced dimeric protein

in solution. Curiously, the dimeric protein was

observed to form the hexamer in the crystal. These

results demonstrate that it remains challenging to pre-

dict oligomeric state in solution from crystal structures

for some systems.

Materials and methods

Production and crystallization of BbPutA

A synthetic gene encoding PutA from Bd. bacteriovorus

HD100 (BbPutA, 982 residues, NCBI RefSeq number NP_

968157.1) with codons optimized for expression in E. coli

was purchased from BIO BASIC Inc. (Markham, Ontario

CA). The gene was subcloned from pUC57 into pKA8H

between NdeI and BamHI sites. The encoded protein has

an N-terminal 8xHis tag that is cleavable by tobacco etch

virus protease (TEVP).

BbPutA was expressed in BL21-AI cells and purified

with affinity chromatography (Ni2+-charged HisTRAP;

GE Healthcare Life Sciences, Pittsburgh, PA, USA) using

protocols described previously for GsPutA [23]. The His

tag was cleaved as described previously for GsPutA [23].

Purified BbPutA was dialyzed into a storage buffer consist-

ing of 50 mM Tris, 125 mM NaCl, 1 mM EDTA, and 1 mM

tris(3-hydroxypropyl)phosphine (THP) at pH 7.5, and then

concentrated to 4 mg�mL�1 using stirred ultrafiltration.

The protein concentration was estimated using the bicin-

choninic acid assay (BCA).

Crystal screening and optimization trials of active

BbPutA produced weakly diffracting crystals, which were

unsuitable for structure determination. Therefore, we pur-

sued crystallization of BbPutA inactivated by the mecha-

nism-based inactivator NPPG (Fig. 4A). Previous studies

have shown that NPPG covalently modifies the FAD and

induces a conformation that resembles the 2-electron

reduced enzyme [23,28,29]. We hypothesized that the

change of conformation might enhance the crystallization

properties of BbPutA. This strategy produced crystals that

diffracted to 2.2 �A resolution.

BbPutA was incubated with NPPG (gift from C. Whit-

man) at a ratio of 1 mg of enzyme per 1 mg of inactivator

for 30 min on ice. The inactivated BbPutA was passed

through a 0.22 lM centrifugal filter device at 4 °C to

remove precipitate. Crystal screening trials were performed

at 20 °C with commercial kits using the microbatch method

with drops formed by mixing 1.5 lL of inactivated BbPutA

(3 mg�mL�1) and 1.5 lL of crystallization reagent. The

drops were covered with Al’s oil (Hampton Research, Aliso

Viejo, CA, USA). Several conditions yielded crystals over-

night. Based on the size and ease of reproducibility, the hit

from reagent 10 of Wizard III (Emerald Biosystems, Bain-

bridge Island, WA, USA) was selected for additional opti-

mization. These efforts produced crystals grown in the

presence of 19% (w/v) polyethylene glycol (PEG) 3350 and

0.25 M KSCN. This crystal form was improved by using

Hampton Index reagents as additives. The base condition

(19% (w/v) PEG 3350, 0.25 M KSCN) was mixed with each

Hampton Index reagent at a ratio of 80 : 20 (base : addi-

tive) and used in microbatch trials. Crystals of equivalent

quality were obtained using Index reagents 45, 72, 73, 76,

79 or 83 as additives. In preparation for cryogenic data col-

lection, the crystals were cryoprotected using 24% (w/v)

PEG 3350, 0.25 M KSCN, and 25% (v/v) ethylene glycol

and plunged into liquid nitrogen. The space group is

P21221 with the unit cell parameters listed in Table 1. The

asymmetric unit contains four protein molecules arranged

as two dimers. The method of Matthews predicts 57% sol-

vent (VM = 2.9 �A3�Da�1) [52].

X-ray diffraction data collection, phasing, and

refinement

Crystals of inactivated BbPutA were analyzed at Advanced

Photon Source beamline 24-ID-E. The data set used for

refinement consisted of 400 frames of data collected with

an oscillation width of 0.25° and detector distance of

300 mm. The data were processed with XDS [53] and SCALA

[54]. Initial phases were determined with molecular replace-

ment as implemented in MOLREP [55] using a search model

derived from the coordinates of GsPutA (47% sequence



http://www.ncbi.nlm.nih.gov/protein/NP_968157.1

http://www.ncbi.nlm.nih.gov/protein/NP_968157.1

identity, Table 2). The structure was refined in PHENIX [56]

and adjusted manually with COOT [57]. The refinement cal-

culations used noncrystallographic symmetry restraints.

The B-factor model consisted of one TLS group per protein

chain and isotropic B-factors for all nonhydrogen atoms.

The model was validated with MOLPROBITY [58,59].

Production of LpPutA and DvPutA

The gene encoding PutA from L. pneumophila subsp. pneu-

mophila (LpPutA, 1054 residues, NCBI RefSeq WP_

010947423.1) was cloned from genomic DNA purchased

from ATCC (catalog number 33152D) and inserted into

pET151. The expressed protein contains an N-terminal

6xHis tag followed the V5 epitope and TEVP cleavage site.

LpPutA was expressed in BL21 DE3 Star cells and purified

with affinity chromatography (Ni2+-charged HisTRAP) and

anion exchange chromatography (HiTrap Q; GE Health-

care) using protocols similar to those described for BjPutA

[60]. The His tag was cleaved as described for BjPutA [60].

The purified protein was dialyzed overnight at 4 °C into a

buffer containing 50 mM Tris-HCl, 50 mM NaCl, 0.5 mM

THP, 5% (v/v) glycerol, and 0.5 mM EDTA at pH 7.5.

The gene encoding PutA from D. vulgaris str. Hildenbor-

ough (DvPutA, 1006 residues, NCBI RefSeq WP_

010940575.1) in the expression plasmid pNIC28-Bsa4 was

obtained from the New York Structural Genomics

Research Consortium. The expressed protein has an N-

terminal 6xHis tag and TEVP cleavage site. DvPutA was

expressed in BL21(DE3)pLysS and purified as described

above for LpPutA. The His tag was not cleaved. The puri-

fied protein was dialyzed overnight at 4 °C into a storage

buffer containing 50 mM Tris-HCl, 50 mM NaCl, 0.5 mM

EDTA, 5% (v/v) glycerol, and 0.5 mM THP at pH 7.5.

Mutagenesis, production, and activity assays of

BjPutA and BjPutA R51E

Wild-type and mutant PutA from Br. japonicum (BjPutA)

in the pKA8H vector were expressed and purified as previ-

ously described [60]. The His tag was removed from both

enzymes as previously described [60]. The R51E mutant

variant of BjPutA was generated from the pKA8H-BjPutA

construct using the QuikChange II XL kit (Agilent, Santa

Clara, CA, USA). The purified proteins were dialyzed over-

night against a storage buffer containing 50 mM Tris (pH

7.8), 50 mM NaCl, 0.5 mM Tris(2-carboxyethyl)phosphine,

and 5% (v/v) glycerol.

Assessment of the coupled PRODH-GSALDH activity

of both wild-type and R51E BjPutA was carried out as pre-

viously described [26]. Briefly, NADH formation was moni-

tored at 340 nm in assays performed at room temperature

in an Epoch 2 plate reader (BioTek, Winooski, VT, USA).

The reaction mixture contained BjPutA or BjPutA R51E

(0.5 lM), proline (2.5–400 mM), coenzyme Q1 (0.1 mM), and

NAD+ (0.2 mM) in a buffer containing 50 mM potassium

phosphate, 25 mM NaCl, and 10 mM MgCl2 at pH 7.5 with

a final reaction volume of 200 lL. Reactions were per-

formed in the presence and absence of NAD+ to correct

for CoQ1 reduction. Linear regression in Origin 2017 was

used to determine rates for final analysis. The rate data

were fit to a substrate inhibition model in Origin 2017.

Analytical ultracentrifugation

Sedimentation velocity experiments were performed in a

Beckman XL-I analytical ultracentrifuge using an An50Ti

rotor at 20 °C. Aliquots of the protein solution and dialysis

buffer (reference buffer) were loaded into a sedimentation

velocity cell, bearing a two-sector charcoal-Epon center-

piece. Prior to centrifugation, the sample was allowed to

equilibrate for 2 h. The sample was then centrifuged at

82 021 g for 300 radial scans at 2-min intervals acquired

using Rayleigh interference optics. Scans 10–300 were used

in the analysis. Sedimentation coefficient, c(s), and molecular

mass, c(M), distributions were generated using SEDFIT [61].

Prior to sedimentation analysis of NPPG-inactivated

BbPutA and BjPutA R51E, each protein was treated with

1 mg NPPG (BOC Sciences, Shirley, NY, USA) per 1 mg

of protein, incubated on ice for 30 min, and then loaded

into the sedimentation cell. The reference for sedimentation

was the respective storage buffer (listed in the purification

protocols above) supplemented with an equal amount of

NPPG used in the inactivation step.

Prior to sedimentation analysis of BbPutA and BjPutA

R51E in the presence of active site ligands, protein samples

were supplemented with 10 mM THFA and 1 mM NAD+

and dialyzed for 4 h with two buffer exchanges in a

Slide-a-lyzer mini-dialysis device (ThermoFisher, Waltham,

MA, USA) against the storage buffer (listed in the purifica-

tion protocol above) supplemented with 10 mM THFA and

1 mM NAD+. The dialysate served as the reference for sed-

imentation.

For all BjPutA wild-type and mutant samples, the fric-

tional ratio was allowed to vary during global fitting. In

the analysis of BbPutA, the frictional ratio was set at 1.75

to account for sample aggregation and precipitation. We

note that the frictional ratio of BjPutA R51E was approxi-

mately 1.75, which is why this frictional ratio was applied

to the BbPutA data.

SAXS

Purified BbPutA was prepared for SAXS by passing it

through a prepacked Superdex-200 10 9 300 mm size

exclusion chromatography (SEC) column (GE Life

Sciences, Pittsburgh, PA, USA) equilibrated with 50 mM

Tris, 125 mM NaCl, 1 mM EDTA, and 1 mM THP at pH

7.5 at a flow rate of 0.5 mL�min�1. The protein eluted as a

single peak that was baseline separated from the void



http://www.ncbi.nlm.nih.gov/protein/WP_010947423.1




volume of the column (Fig. 11). Fractions under the peak

were pooled for SAXS analysis. A sample of the SEC flow-

through was reserved for use in the SAXS background

measurement.

Purified LpPutA and DvPutA were prepared for SAXS

by passing them through the aforementioned SEC column

equilibrated with 50 mM Tris, 500 mM NaCl, and 1 mM

THP at pH 8.0 (flow rate of 0.5 mL�min�1). Each protein

eluted as a single peak that was baseline separated from

the void volume. The protein in the single peak was pooled

and concentrated using a centrifugal concentration device

(30-kDa cutoff) to 8 mg�mL�1 as monitored by the BCA

assay. The SEC equilibration buffer was used for the SAXS

background measurement.

Purified wild-type BjPutA and R51E were prepared for

SAXS by passing them through the aforementioned SEC

column equilibrated with 50 mM Tris (pH 7.8), 50 mM NaCl,

0.5 mM Tris(2-carboxyethyl)phosphine, and 5% (v/v) glyc-

erol (flow rate of 0.5 mL�min�1). Each protein eluted as a

single peak that was baseline separated from the void volume

(Fig. 11). The protein in the single peak was pooled and con-

centrated using a centrifugal concentration device (50-kDa

cutoff). The concentrated protein was dialyzed overnight

using the SEC buffer. The final protein concentration after

dialysis was 7 mg�mL�1 (BCA method). A sample of the dia-

lysate was reserved for the SAXS background measurement.

The protein samples were transferred to 96-well plates

and shipped at 4 °C to the SIBYLS beamline 12.3.1 of the

Advanced Light Source through the mail-in program

[62,63]. Additional SEC steps were not performed at the

beamline. The SAXS intensity data – I(q) vs. q, q = 4psinh/k, where 2h is the scattering angle and k is the X-ray

wavelength in angstroms – were measured at three nominal

protein concentrations in the range of 1–8 mg�mL�1. Note

that scattering intensities for BbPutA were calculated only

at a single protein concentration, 1.8 mg�mL�1. Because of

the low concentration of the BbPutA SEC fractions, data

were collected at a single concentration of 1.8 mg�mL�1.

For BbPutA, DvPutA, and LpPutA, data were collected

for each protein concentration at exposure times of 0.5,

1.0, 3.0, and 6.0 s. For both wild-type BjPutA and the

BjPutA R51E mutant variant, data were collected in shut-

terless mode using a Pilatus detector with a total of 33

evenly spaced images acquired over 10.2 s (0.3 s�frame�1).

The scattering curves collected from the protein samples

were corrected for background scattering using intensity

data collected from the aforementioned reference buffers.

For all except the BjPutA and BjPutA R51E samples,

composite scattering curves were generated with PRIMUS [64]

by scaling and merging the background-corrected low q

region data from the 0.5 s exposure with the high q region

data from the 3.0 s exposure. For both wild-type BjPutA

and BjPutA R51E, composite scattering curves were gener-

ated with PRIMUS by averaging and merging the back-

ground-corrected low q region data from the first three

(0.9 s) exposures with the high q region data from the first

12 (3.6 s) exposures.

The composite SAXS curves were analyzed as follows.

PRIMUS was used to perform Guinier analysis. GNOM was

used to calculate distance distribution functions [65]. FoXS

and MultiFoXS [35] were used to calculate theoretical

SAXS curves from atomic models. For BjPutA and

BbPutA, crystal structures were input to FoXS. For

DvPutA and LpPutA, homology models were generated

using SWISS-MODEL [66], RaptorX [67], and Phyre2 [68].

The LpPutA model was improved with AllosMod-FoXS

[34,69], which added missing residues and allowed for inter-

domain movements. The molecular mass in solution was

determined from SAXS data using the volume of correla-

tion invariant [31] as implemented in Scatter 3.0 [70] and

with the SAXS MoW2 server [32].

DAMMIF [36] was used for shape reconstructions. For

each reconstruction, 50 independent calculations were per-

formed. Two-fold symmetry was enforced during the recon-

structions of the dimeric PutAs (BbPutA, DvPutA,

LpPutA, and BjPutA R51E). Point group 222 symmetry

was enforced during the shape reconstruction of wild-type

BjPutA. The models from DAMMIF were averaged and fil-

tered with DAMAVER [71]. The averaged and filtered dummy

atom models (dammif.pdb) were superimposed onto crystal

structures or homology models with SUPCOMB [72]. The

pdb2vol utility of situs [73] was used to convert dummy

atom models (dammif.pdb) into volumetric maps. The col-

ores utility of situs was used to calculate the correlation

coefficient between atomic models and volumetric maps.

The SAXS data have been deposited in the SASBDB [74]

under the following accession codes: SASDCP3 (BbPutA),

Fig. 11. SEC chromatograms for BbPutA (blue), wild-type BjPutA

(black), and BjPutA R51E (red) obtained with a prepacked

Superdex-200 10 9 300 mm SEC column (GE Life Sciences)

connected to an AKTA pure chromatography instrument (GE Life

Sciences).



SASDCQ3 (DvPutA 1.5 mg�mL�1), SASDCX3 (DvPutA

3.0 mg�mL�1), SASDCY3 (DvPutA 4.5 mg�mL�1),

SASDCR3 (LpPutA 3.0 mg�mL�1), SASDCV3 (LpPutA

5.0 mg�mL�1), SASDCW3 (LpPutA 8.0 mg�mL�1),

SASDCS3 (BjPutA 2.3 mg�mL�1), SASDCT3 (BjPutA

4.7 mg�mL�1), SASDCU3 (BjPutA 7.0 mg�mL�1),

SASDCZ3 (R51E 2.3 mg�mL�1), SASDC24 (R51E

4.7 mg�mL�1), SASDC34 (R51E 7.0 mg�mL�1),

Acknowledgements

Research reported in this publication was supported

by the NIGMS of the National Institutes of Health

under award number R01GM065546. We thank

Kevin Dyer and Katherine Burnett for collecting

SAXS data through the SIBYLS mail-in program.

We thank Jonathon Schuermann for help with X-ray

diffraction data collection and processing. We thank

Chris Whitman and William Johnson, Jr. for provid-

ing the NPPG that was used for crystallization of

BbPutA. We thank Prof Steven Almo and the New

York Structural Genomics Consortium for providing

the DvPutA plasmid. We thank Dina Schneidman for

help with running the AllosMod-FoXS server. Part of

this research was performed at the Advanced Light

Source. The Advanced Light Source is supported by

the Director, Office of Science, Office of Basic Energy

Sciences, of the U.S. Department of Energy under

contract no. DE-AC02-05CH11231. Additional sup-

port for the SYBILS beamline comes from the

National Institute of Health project MINOS

(R01GM105404). Part of this work is based on

research conducted at the Northeastern Collaborative

Access Team beamlines, which are funded by the

National Institute of General Medical Sciences from

the National Institutes of Health (P41 GM103403).

This research used resources of the Advanced Photon

Source, a U.S. Department of Energy (DOE) Office

of Science User Facility operated for the DOE Office

of Science by Argonne National Laboratory under

contract no. DE-AC02-06CH11357.

Conflict of interest

The authors declare that they have no conflicts of

interest with the contents of this manuscript.

Author contributions

DAK, HS, and JJT designed experiments; DAK, HS,

and TAP performed experiments; DAK, HS, and JJT

analyzed data; ML and RD contributed reagents;

DAK and JJT wrote the article.

References

1 Tanner JJ (2008) Structural biology of proline

catabolism. Amino Acids 35, 719–730.2 Phang JM, Liu W, Hancock C & Christian KJ (2012)

The proline regulatory axis and cancer. Front Oncol 2,

60.

3 Phang JM, Hu CA & Valle D (2001) Disorders of

proline and hydroxyproline metabolism. In Metabolic

and Molecular Basis of Inherited Disease (Scriver CR,

Beaudet AL, Sly WS & Valle D, eds), pp. 1821–1838.McGraw Hill, New York, NY.

4 Jacquet H, Raux G, Thibaut F, Hecketsweiler B, Houy

E, Demilly C, Haouzir S, Allio G, Fouldrin G, Drouin

V et al. (2002) PRODH mutations and hyperprolinemia

in a subset of schizophrenic patients. Hum Mol Genet

11, 2243–2249.5 Liu H, Heath SC, Sobin C, Roos JL, Galke BL,

Blundell ML, Lenane M, Robertson B, Wijsman EM,

Rapoport JL et al. (2002) Genetic variation at the

22q11 PRODH2/DGCR6 locus presents an unusual

pattern and increases susceptibility to schizophrenia.

Proc Natl Acad Sci USA 99, 3717–3722.6 Jacquet H, Demily C, Houy E, Hecketsweiler B, Bou J,

Raux G, Lerond J, Allio G, Haouzir S, Tillaux A et al.

(2005) Hyperprolinemia is a risk factor for

schizoaffective disorder. Mol Psychiatry 10, 479–485.7 Willis A, Bender HU, Steel G & Valle D (2008)

PRODH variants and risk for schizophrenia. Amino

Acids 35, 673–679.8 Clelland CL, Read LL, Baraldi AN, Bart CP, Pappas

CA, Panek LJ, Nadrich RH & Clelland JD (2011)

Evidence for association of hyperprolinemia with

schizophrenia and a measure of clinical outcome.

Schizophr Res 131, 139–145.9 Zarse K, Schmeisser S, Groth M, Priebe S, Beuster G,

Kuhlow D, Guthke R, Platzer M, Kahn CR & Ristow M

(2012) Impaired insulin/IGF1 signaling extends life span

by promoting mitochondrial L-proline catabolism to

induce a transient ROS signal. Cell Metab 15, 451–465.10 Lee IR, Lui EY, Chow EW, Arras SD, Morrow CA &

Fraser JA (2013) Reactive oxygen species homeostasis

and virulence of the fungal pathogen Cryptococcus

neoformans requires an intact proline catabolism

pathway. Genetics 194, 421–433.11 Krishnan N, Doster AR, Duhamel GE & Becker DF

(2008) Characterization of a Helicobacter hepaticus

putA mutant strain in host colonization and oxidative

stress. Infect Immun 76, 3037–3044.12 Nakajima K, Inatsu S, Mizote T, Nagata Y, Aoyama

K, Fukuda Y & Nagata K (2008) Possible involvement

of put A gene in Helicobacter pylori colonization in the

stomach and motility. Biomed Res 29, 9–18.13 Berney M, Weimar MR, Heikal A & Cook GM (2012)

Regulation of proline metabolism in mycobacteria and



its role in carbon metabolism under hypoxia. Mol

Microbiol 84, 664–681.14 Cheng Z, Lin M & Rikihisa Y (2014) Ehrlichia

chaffeensis proliferation begins with NtrY/NtrX and

PutA/GlnA upregulation and CtrA degradation

induced by proline and glutamine uptake. MBio 5,

e02141.

15 Tanner JJ & Becker DF (2013) PutA and proline

metabolism. In Handbook of Flavoproteins Volume 1

Oxidases, Dehydrogenases and Related Systems (Hille

R, Miller SM & Palfey BA, eds), pp. 31–56. De

Gruyter, Berlin.

16 Wood JM & Zadworny D (1980) Amplification of the

put genes and identification of the put gene products in

Escherichia coli K12. Can J Biochem 58, 787–796.17 Menzel R & Roth J (1981) Purification of the putA

gene product. J Biol Chem 256, 9755–9761.18 Arentson BW, Sanyal N & Becker DF (2012) Substrate

channeling in proline metabolism. Front Biosci 17, 375–388.

19 Surber MW & Maloy S (1998) The PutA protein of

Salmonella typhimurium catalyzes the two steps of

proline degradation via a leaky channel. Arch Biochem

Biophys 354, 281–287.20 Srivastava D, Schuermann JP, White TA, Krishnan N,

Sanyal N, Hura GL, Tan A, Henzl MT, Becker DF &

Tanner JJ (2010) Crystal structure of the bifunctional

proline utilization A flavoenzyme from Bradyrhizobium

japonicum. Proc Natl Acad Sci USA 107, 2878–2883.21 Luo M, Christgen S, Sanyal N, Arentson BW, Becker

DF & Tanner JJ (2014) Evidence that the C-terminal

domain of a type B PutA protein contributes to

aldehyde dehydrogenase activity and substrate

channeling. Biochemistry 53, 5661–5673.22 Moxley MA, Sanyal N, Krishnan N, Tanner JJ &

Becker DF (2014) Evidence for hysteretic substrate

channeling in the proline dehydrogenase and delta1-

pyrroline-5-carboxylate dehydrogenase coupled reaction

of proline utilization A (PutA). J Biol Chem 289, 3639–3651.

23 Singh H, Arentson BW, Becker DF & Tanner JJ (2014)

Structures of the PutA peripheral membrane

flavoenzyme reveal a dynamic substrate-channeling

tunnel and the quinone-binding site. Proc Natl Acad Sci

USA 111, 3389–3394.24 Luo M, Gamage TT, Arentson BW, Schlasner KN,

Becker DF & Tanner JJ (2016) Structures of proline

utilization A (PutA) reveal the fold and functions of the

aldehyde dehydrogenase superfamily domain of

unknown function. J Biol Chem 291, 24065–24075.25 Korasick D, Gamage TT, Christgen S, Stiers KM,

Beamer LJ, Henzl MT, Becker DF & Tanner JJ (2017)

Structure and characterization of a class 3B proline

utilization A: ligand-induced dimerization and

importance of the C-terminal domain for catalysis.

J Biol Chem 292, 9652–9665.26 Arentson BW, Luo M, Pemberton TA, Tanner JJ &

Becker DF (2014) Kinetic and structural

characterization of tunnel-perturbing mutants in

Bradyrhizobium japonicum proline utilization A.

Biochemistry 53, 5150–5161.27 Singh RK, Larson JD, Zhu W, Rambo RP, Hura GL,

Becker DF & Tanner JJ (2011) Small-angle X-ray

scattering studies of the oligomeric state and quaternary

structure of the trifunctional proline utilization A

(PutA) flavoprotein from Escherichia coli. J Biol Chem

286, 43144–43153.28 Srivastava D, Zhu W, Johnson WH Jr, Whitman CP,

Becker DF & Tanner JJ (2010) The structure of the

proline utilization a proline dehydrogenase domain

inactivated by N-propargylglycine provides insight into

conformational changes induced by substrate binding

and flavin reduction. Biochemistry 49, 560–569.29 White TA, Johnson WH Jr, Whitman CP & Tanner JJ

(2008) Structural basis for the inactivation of Thermus

thermophilus proline dehydrogenase by N-

propargylglycine. Biochemistry 47, 5573–5580.30 Luo M, Arentson BW, Srivastava D, Becker DF &

Tanner JJ (2012) Crystal structures and kinetics of

monofunctional proline dehydrogenase provide insight

into substrate recognition and conformational changes

associated with flavin reduction and product release.

Biochemistry 51, 10099–10108.31 Rambo RP & Tainer JA (2013) Accurate assessment of

mass, models and resolution by small-angle scattering.

Nature 496, 477–481.32 Fischer H, de Oliveira Neto M, Napolitano HB,

Polikarpov I & Craievich AF (2010) Determination of

the molecular weight of proteins in solution from a

single small-angle X-ray scattering measurement on a

relative scale. J Appl Crystallogr 43, 101–109.33 Krissinel E & Henrick K (2007) Inference of

macromolecular assemblies from crystalline state. J Mol

Biol 372, 774–797.34 Schneidman-Duhovny D, Hammel M & Sali A (2010)

FoXS: a web server for rapid computation and

fitting of SAXS profiles. Nucleic Acids Res 38, W540–W544.

35 Schneidman-Duhovny D, Hammel M, Tainer JA & Sali

A (2016) FoXS, FoXSDock and MultiFoXS: single-

state and multi-state structural modeling of proteins

and their complexes based on SAXS profiles. Nucleic

Acids Res 44, W424–W429.

36 Franke D & Svergun DI (2009) DAMMIF, a program

for rapid ab-initio shape determination in small-angle

scattering. J Appl Crystallogr 42, 342–346.37 Zhu W, Gincherman Y, Docherty P, Spilling CD &

Becker DF (2002) Effects of proline analog binding on



the spectroscopic and redox properties of PutA. Arch

Biochem Biophys 408, 131–136.38 Zhang M, White TA, Schuermann JP, Baban BA,

Becker DF & Tanner JJ (2004) Structures of the

Escherichia coli PutA proline dehydrogenase domain in

complex with competitive inhibitors. Biochemistry 43,

12539–12548.39 Goodsell DS & Olson AJ (2000) Structural symmetry

and protein function. Annu Rev Biophys Biomol Struct

29, 105–153.40 Levy ED & Teichmann S (2013) Structural,

evolutionary, and assembly principles of protein

oligomerization. Prog Mol Biol Transl Sci 117, 25–51.41 Marsh JA & Teichmann SA (2015) Structure,

dynamics, assembly, and evolution of protein

complexes. Annu Rev Biochem 84, 551–575.42 Christensen EM, Patel SM, Korasick DA, Campbell

AC, Krause KL, Becker DF & Tanner JJ (2017)

Resolving the cofactor binding site in the proline

biosynthetic enzyme human pyrroline-5-carboxylate

reductase 1. J Biol Chem 292, 7233–7243.43 Rodriguez-Zavala JS & Weiner H (2002) Structural

aspects of aldehyde dehydrogenase that influence dimer-

tetramer formation. Biochemistry 41, 8229–8237.44 Korasick DA, Tanner JJ & Henzl MT (2017) Impact of

disease-linked mutations targeting the oligomerization

interfaces of aldehyde dehydrogenase 7A1. Chem Biol

Interact. https://doi.org/10.1016/j.cbi.2017.01.002

45 Veenhoff LM, Heuberger EH & Poolman B (2002)

Quaternary structure and function of transport

proteins. Trends Biochem Sci 27, 242–249.46 Perica T, Marsh JA, Sousa FL, Natan E, Colwell LJ,

Ahnert SE & Teichmann SA (2012) The emergence of

protein complexes: quaternary structure, dynamics and

allostery. Colworth Medal Lecture. Biochem Soc Trans

40, 475–491.47 Cornish-Bowden A (2014) Understanding allosteric and

cooperative interactions in enzymes. FEBS J 281, 621–632.

48 Jaffe EK (2005) Morpheeins – a new structural

paradigm for allosteric regulation. Trends Biochem Sci

30, 490–497.49 Bogan AA & Thorn KS (1998) Anatomy of hot spots

in protein interfaces. J Mol Biol 280, 1–9.50 Moreira IS, Fernandes PA & Ramos MJ (2007) Hot

spots – a review of the protein-protein interface

determinant amino-acid residues. Proteins 68, 803–812.51 Luo M, Singh RK & Tanner JJ (2013) Structural

determinants of oligomerization of delta(1)-pyrroline-5-

carboxylate dehydrogenase: identification of a

hexamerization hot spot. J Mol Biol 425, 3106–3120.52 Matthews BW (1968) Solvent content of protein

crystals. J Mol Biol 33, 491–497.53 Kabsch W (2010) XDS. Acta Crystallogr D 66, 125–

132.

54 Evans P (2006) Scaling and assessment of data quality.

Acta Crystallogr D 62, 72–82.55 Vagin A & Teplyakov A (2000) An approach to multi-

copy search in molecular replacement. Acta Crystallogr

D 56, 1622–1624.56 Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis

IW, Echols N, Headd JJ, Hung LW, Kapral GJ,

Grosse-Kunstleve RW et al. (2010) PHENIX: a

comprehensive Python-based system for

macromolecular structure solution. Acta Crystallogr D

66, 213–221.57 Emsley P, Lohkamp B, Scott WG & Cowtan K (2010)

Features and development of Coot. Acta Crystallogr D

66, 486–501.58 Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral

GJ, Wang X, Murray LW, Arendall WB III, Snoeyink

J, Richardson JS et al. (2007) MolProbity: all-atom

contacts and structure validation for proteins and

nucleic acids. Nucleic Acids Res 35, W375–W383.

59 Chen VB, Arendall WB III, Headd JJ, Keedy DA,

Immormino RM, Kapral GJ, Murray LW, Richardson

JS & Richardson DC (2010) MolProbity: all-atom

structure validation for macromolecular

crystallography. Acta Crystallogr D 66, 12–21.60 Schuermann JP, White TA, Srivastava D, Karr DB &

Tanner JJ (2008) Three crystal forms of the bifunctional

enzyme proline utilization A (PutA) from Bradyrhizobium

japonicum. Acta Crystallogr F 64, 949–953.61 Schuck P (2000) Size-distribution analysis of

macromolecules by sedimentation velocity

ultracentrifugation and lamm equation modeling.

Biophys J 78, 1606–1619.62 Hura GL, Menon AL, Hammel M, Rambo RP, Poole

FL II, Tsutakawa SE, Jenney FE Jr, Classen S, Frankel

KA, Hopkins RC et al. (2009) Robust, high-throughput

solution structural analyses by small angle X-ray

scattering (SAXS). Nat Methods 6, 606–612.63 Classen S, Hura GL, Holton JM, Rambo RP, Rodic

I, McGuire PJ, Dyer K, Hammel M, Meigs G,

Frankel KA et al. (2013) Implementation and

performance of SIBYLS: a dual endstation small-

angle X-ray scattering and macromolecular

crystallography beamline at the Advanced Light

Source. J Appl Crystallogr 46, 1–13.64 Konarev PV, Volkov VV, Sokolova AV, Koch MHJ &

Svergun DI (2003) PRIMUS: a windows PC-based

system for small-angle scattering data analysis. J Appl

Crystallogr 36, 1277–1282.65 Svergun D (1992) Determination of the regularization

parameter in indirect-transform methods using

perceptual criteria. J Appl Crystallogr 25, 495–503.66 Biasini M, Bienert S, Waterhouse A, Arnold K, Studer

G, Schmidt T, Kiefer F, Gallo Cassarino T, Bertoni M,

Bordoli L et al. (2014) SWISS-MODEL: modelling

protein tertiary and quaternary structure using



https://doi.org/10.1016/j.cbi.2017.01.002

evolutionary information. Nucleic Acids Res 42, W252–W258.

67 Kallberg M, Margaryan G, Wang S, Ma J & Xu J (2014)

RaptorX server: a resource for template-based protein

structure modeling. Methods Mol Biol 1137, 17–27.68 Kelley LA & Sternberg MJ (2009) Protein structure

prediction on the Web: a case study using the Phyre

server. Nat Protoc 4, 363–371.69 Weinkam P, Pons J & Sali A (2012) Structure-based

model of allostery predicts coupling between distant

sites. Proc Natl Acad Sci USA 109, 4875–4880.70 Rambo RP (2015) Scatter in https://bl1231.als.lbl.gov/

scatter/.

71 Volkov VV & Svergun DI (2003) Uniqueness of

ab initio shape determination in small-angle scattering.

J Appl Crystallogr 36, 860–864.72 Kozin MB & Svergun DI (2001) Automated matching

of high- and low-resolution structural models. J Appl

Crystallogr 34, 33–41.

73 Wriggers W (2010) Using situs for the integration of

multi-resolution structures. Biophys Rev 2, 21–27.74 Valentini E, Kikhney AG, Previtali G, Jeffries CM &

Svergun DI (2015) SASBDB, a repository for biological

small-angle scattering data. Nucleic Acids Res 43,

D357–D363.

75 Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K,

Li W, Lopez R, McWilliam H, Remmert M, Soding J

et al. (2011) Fast, scalable generation of high-quality

protein multiple sequence alignments using Clustal

Omega. Mol Syst Biol 7, 539.

76 Dereeper A, Guignon V, Blanc G, Audic S, Buffet S,

Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot

M et al. (2008) Phylogeny.fr: robust phylogenetic

analysis for the non-specialist. Nucleic Acids Res 36,

W465–W469.



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Biophysical investigation of type A PutAs reveals a...

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