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J. Bacteriol. manuscript March 24, 2010

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S-ADENOSYLMETHIONINE-BINDING PROPERTIES OF A BACTERIAL 2

PHOSPHOLIPID N-METHYLTRANSFERASE 3

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Meriyem Aktas1, Jan Gleichenhagen1, Raphael Stoll2 and Franz Narberhaus1* 5

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Microbial Biology1 and Biomolecular NMR2, Ruhr-University Bochum, Germany 7

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Running title: SAM binding by Agrobacterium PmtA 9

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Keywords: Agrobacterium tumefaciens, phospholipid biosynthesis, phospholipid N-11

methyltransferase, phosphatidylcholine, S-adenosylmethionine methyltransferase 12

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* Corresponding author Franz Narberhaus, Lehrstuhl für Biologie der 14

Mikroorganismen, Universitätsstrasse 150, NDEF 06/783, Ruhr-Universität Bochum, 15

D-44780 Bochum, Germany, Tel. 49 (234) 32 23100, Fax. 49 (234) 32 14620, E-16

Mail: [email protected]

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.01539-10 JB Accepts, published online ahead of print on 20 May 2011

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The presence of the membrane lipid phosphatidylcholine (PC) in the 1

bacterial membrane is critically important for many host-microbe interactions. 2

The phospholipid N-methyltransferase PmtA from the plant pathogen 3

Agrobacterium tumefaciens catalyzes the formation of PC by a three-step 4

methylation of phosphatidylethanolamine via monomethyl-5

phosphatidylethanolamine and dimethylphosphatidylethanolamine. The 6

methyl group is provided by S-adenosylmethionine (SAM), which is converted 7

to S-adenosylhomocysteine (SAH) during transmethylation. Despite the 8

biological importance of bacterial phospholipid N-methyltransferases, little is 9

known about amino acids critical for binding to SAM or phospholipids and 10

catalysis. Alanine substitutions in the predicted SAM-binding residues E58, 11

G60, G62 and E84 in A. tumefaciens PmtA dramatically reduced SAM binding 12

and enzyme activity. Homology modelling of PmtA satisfactorily explained the 13

mutational results. The enzyme is predicted to exhibit a consensus topology of 14

the SAM-binding fold consistent with cofactor interaction as seen in most 15

structurally characterized SAM-methyltransferases. NMR-titration experiments 16

and 14C-SAM-binding studies revealed binding constants for SAM and SAH in 17

the low micromolar range. Our study provides first insights into structural 18

features and SAM binding of a bacterial phospholipid N-methyltransferase. 19


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Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotes 1

and the key building block of membrane bilayers. As a major source of lipid second 2

messenger it plays an important role in signal transduction (15). Although most 3

prokaryotes lack PC, it is present in substantial amounts in membranes of rather 4

diverse bacteria. It has been estimated that about 10 % of all bacterial species 5

possess PC (47). Intriguingly, many of these bacteria interact with eukaryotes and a 6

number of pathogenic and symbiotic PC-containing bacteria like 7

Agrobacterium tumefaciens, Bradyrhizobium japonicum, Brucella abortus and 8

Legionella pneumophila require PC for an efficient interaction with their respective 9

hosts (2, 11, 12, 28, 33, 52). Two PC biosynthesis pathways operate in prokaryotes. 10

In the PC synthase (Pcs) pathway, choline is directly condensed with CDP-11

diacylglycerol (CDP-DAG) to form PC in a reaction catalyzed by Pcs. In the 12

methylation pathway, PC is formed by three consecutive methylations of 13

phosphatidylethanolamine (PE) via the intermediates monomethyl- (MMPE) and 14

dimethylphosphatidylethanolamine (DMPE). Depending on the bacterial species, the 15

transmethylation reactions are catalyzed by one or more phospholipid N-16

methyltransferases (Pmt) using S-adenosylmethionine (SAM) as a methyldonor (18, 17

28, 47). Many eukaryotes also use a methylation pathway for PC formation (8, 13, 18

51). However, their enzymes differ from bacterial Pmt enzymes in sequence and 19

structure. 20

Phospholipid N-methyltransferases belong to the SAM-dependent methyltransferase 21

(SAM-MTase) family. Members of this diverse class of enzymes catalyze the transfer 22

of the methyl group from the ubiquitous cofactor SAM to proteins, nucleic acids, 23

lipids or small molecules (30, 32, 42). Consistent with these structurally diverse 24

substrates, the substrate-recognition domain in the C-terminus of SAM-MTases is 25

highly variable (27, 30). On the other hand, SAM-MTases contain a conserved N-26


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terminal SAM-binding fold comprised of a central seven-stranded ß-sheet, flanked by 1

three α-helices on each side. Except for a few key residues, amino acids involved in 2

SAM binding are diverse. So far, about ten sequence motifs (SAM-I to X) important 3

for SAM binding and catalysis have been described (27, 29). The only highly 4

conserved residues are the glycine-rich sequence E/D-X-G-X-G-X-G (SAM-I) and an 5

acidic loop between the second β-strand and the following α-helix (SAM-II). Motif-I, 6

II, III and X are primarily responsible for SAM binding, and motifs-IV, VI, and VIII in 7

the active site are involved in catalysis (10, 42). 8

PC in the plant pathogen A. tumefaciens is synthesized via the Pcs and the 9

methylation pathway. In the latter, all three methylation steps are catalyzed by a 10

single Pmt enzyme called PmtA (24, 52). An A. tumefaciens strain lacking both PC 11

biosynthesis pathways is unable to elicit plant tumors (52). Recently, we produced 12

recombinant PmtA in Escherichia coli and characterized the enzyme properties in 13

vitro (1, 26). PmtA acts as a monomeric enzyme of 22.3 kDa, and is inhibited by the 14

end products PC and S-adenosylhomocysteine (SAH). SAM binding strictly depends 15

on the presence of phospholipid substrate (1). In a step towards understanding the 16

catalytic mechanism of bacterial Pmt enzymes we measured SAM-binding affinities 17

and constructed a series of PmtA point mutants in residues thought to be critical for 18

SAM binding. A three-dimensional homology model agrees well with the 19

mutagenesis data. 20


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MATERIAL AND METHODS 1

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Materials. 3-sn-phosphatidyl-ethanolamine was purchased from Sigma Aldrich. S-3

[methyl-14C]adenosyl-L-methionine (1.806 GBq; 48.8 mCi/mmol) was obtained from 4

Hartmann Analytic. HAWP 02500 filters for SAM-binding assays were purchased 5

from Millipore and Hybond-C extra membranes for protein-lipid overlay assays from 6

Amersham. The HPTLC silica gel 60 plates were from Merck. All other reagents 7

were of the highest standard commercially available. 8

Bacterial strains and growth conditions. Bacterial strains and plasmids used in 9

the present study are listed in Table 1. E. coli cells were grown at 37°C in Luria-10

Bertani (LB) broth or on LB agar plates supplemented with kanamycin (Km) at a final 11

concentration of 50 µg/ml. E. coli DH5α was used as host for all cloning procedures. 12

E. coli BL21(DE3) served as host for overproduction of PmtA variants. 13

Site-directed mutagenesis. To generate PmtA E58A, G60A, P61A, G62A, G64A, 14

E84A, D106A, D106E and G162A variants site-directed mutagenesis was conducted 15

using the QuikChange® mutagenesis kit (Stratagene) following the supplier’s 16

protocol. The sequence of primers used in this study is presented in supplementary 17

Table 1. The vector pBO832 (pET28b containing wild-type (wt) pmtA; (1)) was 18

subjected to site-directed mutagenesis. Mutated pmtA variants were verified by 19

sequencing. 20

Expression and purification of PmtA proteins in E. coli. E. coli BL21(DE3) 21

carrying wt or mutated pmtA in pET28b was cultivated in LB medium containing 22

kanamycin at 37°C until the OD600 nm reached a value between 0.5 and 0.8. Then, 23

synthesis of PmtA was induced by addition of isopropyl-β-D-thiogalactopyranoside 24

(IPTG) to a final concentration of 0.4 mM and the cultures were incubated for 25

another 2 h at 30°C. 1 ml culture of the BL21 was harvested by centrifugation for 26


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SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and 2 ml of the cultures for 1

lipid analysis via thin-layer chromatography (TLC). For SDS-PAGE cell pellets were 2

resuspended in 1x SDS-loading buffer according to the OD600 nm (OD600 nm 1 ≈ 100 µl 3

1x SDS loading buffer) and boiled for 10 min. 10 µl of each sample were separated 4

on 12.5 % SDS-polyacrylamide gels and the proteins were stained with Coomassie 5

Blue. 6

PmtA wt and mutant proteins were purified as described previously (1). 7

Analysis of PmtA reaction products via thin-layer chromatography. The lipid 8

composition of E. coli strains producing PmtA derivatives was determined via TLC. 9

Cells were cultivated as mentioned above, 2 ml cultures were harvested by 10

centrifugation, washed with 500 µl water and resuspended in 100 µl water. The lipids 11

were extracted according to Bligh and Dyer (5), separated by one-dimensional TLC 12

using HPTLC silica gel 60 plates (Merck) and stained with Cu2SO4 solution (300 mM 13

copper(II)-sulfate-pentahydrate; 8.5 % (v/v) phosphoric acid). As running solvent n-14

propanol/propionate/chloroform/water (3:2:2:1) was used. 15

Circular dichroism (CD) spectroscopy. The CD spectra of recombinant PmtA 16

proteins were recorded 10 times between 190 and 320 nm on a JASCO 715 17

spectropolarimeter at 20°C in 50 mM potassium phosphate buffer, pH 8. PmtA 18

thermostability was measured in duplicate with 10 µM of enzyme over a temperature 19

range from 5° to 80°C in increments of 5°C. The final spectra obtained were the 20

average of the 10 scans, normalized against buffer. Analyzes were performed in 21

duplicate using 10 µM of enzyme. 22

The wt PmtA CD-spectrum was analyzed using K2D2 (17, 36) and CDSSTR (53) 23

algorithms. The experimentally estimated secondary structure content of PmtA was 24

compared to the predicted secondary structure calculated via GORIV (16) and PHD 25

(39) and to the homology model with the program Stride (20). 26


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Protein lipid overlay assay. Protein lipid overlay assays were carried out as 1

described previously (1). 1 µl of a 14 nmol/µl PE solution or 2 µl of a A. tumefaciens 2

lipid solution in a mixture of chloroform-methanol-water (1:2:0.8) (extracted from a 2 3

ml A. tumefaciens cell culture according to Bligh and Dyer (5) was spotted onto 4

Hybond-C extra membrane strips and air-dried for 1 h at room temperature. 5

Membranes were blocked in blocking buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 6

0.1 % (v/v) Tween-20, 2 % (w/v) fatty acid-free BSA) for 1 h at room temperature and 7

incubated with 300 µg of recombinant PmtA protein variants in 5 ml blocking buffer 8

for 2 h at room temperature. Membranes were washed six times for 5 min in TBST 9

buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.1 % (v/v) Tween-20) and His-10

tagged proteins bound to the lipids were detected with an anti-Penta-His horse 11

radish peroxidase (HRP) coupled antibody (Qiagen) and a chemiluminescence 12

(ECL) Western blotting detection system according to the manufacturer’s instructions 13

(GE Healthcare). 14

Radioligand-binding studies. SAM-binding assays were carried out as described 15

previously (1). Briefly, 10 µM of recombinant PmtA protein was incubated with 16

100 µM phosphatidylethanolamine liposomes (100 nm) and 0-200 µM of S-[methyl-17

14C]adenosyl-L-methionine (48.8 mCi/mmol) in binding buffer (KH2PO4 50 mM, 18

pH 8.0, adjusted with KOH) for 10 min at 30°C (total assay volume 50 µl). Binding 19

assay mixtures were passed over HAWP 02500 filters on a filtration funnel, and 20

unbound S-[methyl-14C]adenosyl-L-methionine was removed by washing four times 21

with 300 µl of binding buffer. Bound S-[methyl-14C]adenosyl-L-methionine was 22

quantified by liquid scintillation spectrometry (Beckman Counter, LS-6000 TA). The 23

Michaelis-Menten equation was used for calculation of SAM-binding affinities of wt 24

and mutated PmtA enzymes. The data were fitted by non-linear regression using 25

SigmaPlot 9.0. 26


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NMR spectroscopy. All spectra were recorded at 298 K and pH 8.0 on Bruker 1

DRX600 and spectrometer equipped with a pulsed field gradient and a triple 2

resonance probehead. Water suppression in experiments recorded on samples in 3

H2O was achieved by incorporation of a Watergate sequence into the various pulse 4

sequences (6, 14, 45). The binding studies were essentially performed as previously 5

published (4, 35, 37, 44, 48, 49). Briefly, 125 µM PmtA was incubated with 250 µM 6

PE-liposomes and 5% D2O in binding buffer (KH2PO4 50 mM, pH 8.0) in a reaction 7

volume of 500 µl for the 1H-titration experiments with SAM and SAH. Then, up to 8

200 µM of either SAM or SAH were titrated to this mixture and 1H-NMR spectra were 9

recorded. For the titration with SAM, the assay was coupled to the activity of 1 µM of 10

SAH-nucleosidase (EC 3.2.2.9, GBiosciences). Conversion of SAH, which is formed 11

during PmtA action, to S-ribosylhomocysteine by the SAH-nucleosidase prevented 12

the accumulation of SAH and its competition with SAM binding (1). 13

Development of a homology model for PmtA. The three-dimensional structure of 14

PmtA was predicted by the threading method using the I-TASSER online server (40, 15

54, 55). Protein structures with following PDB-ID: 3fuxC, 3bkwA, 3futA, 1zg9A, 16

1qaqA, 1gyrA, 3ggdA and 3futA were chosen by I-TASSER as the templates in the 17

modelling procedure. This server produced five possible models for PmtA. The first 18

model with the best quality of prediction (C-score: -0.79, TM-score: 0.61 ± 0.14 and 19

RMSD: 7.0 ± 4.14 Å) was used here. 20


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RESULTS 1

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Prediction of key residues for SAM binding. PmtA catalyzes a series of SAM-3

dependent methylation reactions to produce PC and SAH. In an attempt to define 4

residues in PmtA with a critical role in SAM binding, a multiple sequence alignment 5

of PmtA with other sinorhizobial Pmt enzymes was generated. The SAM-binding 6

motif-I (SAM-I) characterized by an acidic residue and a glycine rich stretch (D/E-X-7

G-X-G-X-G) is highly conserved in all Pmt enzymes and easily discernible (Fig. 1). 8

Regardless of the substrate, this motif is characteristic for all SAM-dependent 9

methyltransferases and is critical for SAM binding (23, 27, 47). In contrast, an 10

unambiguous assignment of other SAM-binding motifs is not plausible without 11

structural guidance due to the sequence heterogeneity in these regions. On the 12

basis of the secondary structure prediction of PmtA (I-TASSER; (40, 54)) we were 13

able to predict two additional sequence motifs (SAM-II and III, Fig. 1) in 14

A. tumefaciens PmtA. Motif-II includes the predicted β-strand 2 and the adjacent 15

turn. An acidic residue, E84 in A. tumefaciens PmtA, is common at the C-terminus 16

of this strand (27). Motif-III is characterized by an acidic residue close to the C-17

terminus of β-strand 3 (27). The conserved D106 in PmtA satisfies the motif-III 18

criteria. 19

Effect of alanine substitutions of putative SAM-motif residues on PmtA 20

activity. In order to investigate the role of the putative SAM-binding residues, we 21

generated seven PmtA derivatives with single alanine substitutions in the predicted 22

SAM motifs described above (Fig. 1). As a control, we constructed a PmtA mutant 23

containing an alanine substitution outside of the predicted SAM motifs (G162A). The 24

effect of these mutations on PmtA activity was analyzed in E. coli BL21(DE3). All 25

proteins were heterologously expressed and successful production was confirmed by 26


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SDS-PAGE (Fig 2A). G64A and D106A were completely insoluble and thus inactive 1

(data not shown). A less severe mutation of D106 to D106E still rendered the protein 2

insoluble (data not shown) suggesting that this residue is critical for proper protein 3

folding. 4

PmtA activity was assayed via TLC of membrane lipids. E. coli is unable to produce 5

PC or any other methylated phospholipids (Fig. 2B, lane V). As reported previously 6

(26), MMPE, DMPE and PC were synthesized when wt PmtA was expressed 7

(Fig. 2B, lane WT). PmtA-P61A and G162A were as active as wt PmtA suggesting 8

that these amino acids are not critical for enzyme activity. G60A and G62A produced 9

only traces of MMPE and no DMPE and PC. E58A and E84A were completely 10

inactive indicating an essential role of these amino acids for PmtA activity. 11

PmtA variants are properly folded. In case soluble proteins were obtained, the 12

mutant proteins were purified according to the procedures previously established for 13

the wt enzyme (1). Their structural integrity was assessed using CD spectroscopy. 14

The spectrum of wt PmtA showed a minimum around 210 nm suggestive of an 15

extensive helical content (Fig. 3A). Overall, the CD profiles of PmtA-E58A, G60A, 16

P61A, G62A, E84A and G162A were similar to that of the wt protein indicating that 17

the mutated PmtA proteins were structurally intact. 18

As a second line of evidence for normally folded proteins we compared their thermal 19

unfolding by CD spectroscopy. Wt PmtA exhibited a Tm of 35.9 C (Fig. 3B) and the 20

six mutated proteins were comparable with wt PmtA having a melting point around 21

35°C (Fig. 3C). 22

The programs CDSSTR and K2D2 were used to calculate the secondary structure 23

content of wt PmtA from the CD spectrum. The estimated α-helical (39-42 %) and β-24

sheet content (~ 11 %) agree well with both the predicted secondary structure of the 25

PmtA amino acid sequence and the homology model structure (Table 2). 26


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Predicted SAM-binding residues are required for SAM binding. To analyze 1

whether the functional defect of the putative SAM-binding motif mutants in vivo 2

(Fig. 2B) was due to a deficiency in SAM binding, we tested their SAM-binding 3

capacity by filter binding assays using 14C-SAM. Consistent with almost wt-like 4

activities in E. coli (Fig. 2B), PmtA-P61A and G162A bound SAM efficiently in vitro 5

(Fig. 4A). The poor enzyme activity of G62A and G60A (Fig. 2B) was reflected by 6

residual SAM binding (Fig. 4A). The inactive E84A enzyme was completely unable to 7

bind SAM. These results suggest a critical role of amino acids E58, G60, G62 and 8

E84 in SAM binding by the methyltransferase. 9

Since PmtA binds SAM only in the presence of one of its phospholipid substrates (1), 10

we needed to exclude that the observed defects in SAM binding were caused by 11

impaired lipid binding. Commercially available PE or total lipids extracted from 12

A. tumefaciens were spotted on a nitrocellulose membrane. Purified His-tagged 13

PmtA variants were incubated with the lipid-displaying membrane and bound 14

proteins were detected with antisera raised against the His-tag. All PmtA derivatives 15

were able to bind both, PE and Agrobacterium total lipids. Consistent with previous 16

findings (1), PmtA binding to the Agrobacterium lipid mixture was stronger than to PE 17

alone (Fig. 4B). 18

Determination of binding constants of PmtA derivatives for SAM by 14C-SAM 19

titration experiments. To determine equilibrium SAM binding constants (KD) for 20

PmtA and its derivatives, we performed 14C-SAM titration experiments in the 21

presence of PE as substrate (Fig. 5). Wt PmtA bound SAM with an affinity in the low 22

micromolar range (~ 25 µM, Fig. 5A). Consistent with their in vivo and in vitro 23

activities, PmtA-P61A and G162A had a similar KD (~ 22 and ~ 30 µM, respectively; 24

Fig. 5B). Since E58A, G60A and G62A bound only marginal amounts of SAM, a KD 25


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calculation for theses PmtA variants was not possible (Fig. 5C). As mentioned 1

above, E84A was completely incapable of binding SAM (Fig. 4 and 5C). 2

Determination of SAM and SAH binding constants for PmtA using 1H NMR-3

titration experiments. A 1D 1H-NMR-spectrum of 125 µM wt PmtA of 4

A. tumefaciens in the presence of 250 µM PE-liposomes in a 50 mM potassium 5

phosphate buffer at pH 8.0 (including 5% D2O) was recorded at 298 K and 600 MHz 6

in order to check the structural integrity of wt PmtA (Fig. 6A). The dispersion of the 7

proton resonances in the aliphatic and amide region clearly shows that the protein 8

adopts a folded conformation. 9

The NMR spectroscopy provided another option for determining the affinity constants 10

of PmtA for SAM and its inhibitor SAH (1). Since SAM binding is impossible in the 11

absence of lipid substrate (1), the NMR-titration experiments were performed in the 12

presence of PE. SAH nucleosidase was added to SAM titration experiments to 13

prevent accumulation of SAH and its competition with SAM binding (see 14

Experimental procedures). Addition of increasing amounts of SAM to PmtA resulted 15

in a decrease in the peak intensities between 0.2 ppm and 0.1 ppm (Fig. 6B, left). 16

With increasing amounts of SAH titrated to the protein sample, a new peak appeared 17

between -0.6 ppm and -0.5 ppm (Fig. 6B, right). The changes of the peak intensities 18

or area were plotted against the SAM/SAH concentration and the binding constants 19

were determined as previously published (21, 22, 25). We were able to estimate a 20

KD in the micromolar range for SAM (~ 91 µM, Fig. 7A) and SAH (~ 25 µM; Fig. 7B). 21


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DISCUSSION 1

In a number of symbiotic or pathogenic bacteria, including the plant pathogen 2

A. tumefaciens, PC biosynthesis is essential for virulence (2, 11, 12, 33, 52). Despite 3

the importance of PC for bacterial fitness, biofilm formation and host-microbe 4

interaction (26, 52), a detailed knowledge about the structural features of bacterial 5

PC biosynthesis enzymes is missing. Here, we studied SAM binding of a PmtA 6

enzymes that catalyses all three methylation reactions from PE to PC (1). Using 7

recombinant protein, we determined binding affinities in the low micromolar range by 8

two independent assays. The 14C-SAM binding assays revealed a KD of 25 µM. This 9

is almost identical to the value determined for the PE N-methyltransferase from rat 10

liver (~30 µM) (38, 43). It is within the range between 0.1 and 30 µM that has 11

previously been reported for diverse SAM-dependent MTases (3, 31, 41). The KD 12

calculated from NMR titration experiments with PmtA was three to four-fold higher 13

(~ 91 µM). This is mostly likely due to residual SAH product that had not been 14

converted by the SAH nucleosidase present in the assay. The determined KD for 15

SAH (~ 25 µM) supports the observation that it is able to compete with SAM for the 16

same binding site (1). 17

Site-directed point mutations of PmtA provided first insights into the mechanism of 18

SAM binding. The conserved topology of the SAM-binding fold in SAM-MTases 19

suggested three putative SAM-binding motifs (SAM-I; II and III) in A. tumefaciens 20

PmtA (Fig. 1). Amino acids E58, G60, P61, G62, G64 (motif-I), E84 (motif-II), and 21

D106 (motif-III) were predicted to form the SAM-binding pocket and were therefore 22

mutated to alanine. 23

E58A, G60A, G62A and E84A were hardly able to convert PE to PC. Their inactivity 24

did not result from failure to fold but from impaired SAM binding. The hypothesis that 25

these amino acids form the SAM-binding pocket is supported by a homology model 26


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of PmtA. All four residues are in close proximity in the three-dimensional structure 1

(Fig. 8A) and may interact directly with SAM (Fig. 8B). The typical SAM-MTase fold 2

(30) consists of a central ß-sheet sandwiched by α-helices (Fig. 8A). The central ß-3

sheet is composed of five parallel ß-strands and an anti-parallel ß-strand with a 4

topological organization of (6↓5↑4↑1↑2↑3). Three of the helices (αE, αF, αG) on one 5

side of the sheet and three helices (αB, αC, αD) on the other side create a α/ß/α 6

secondary superstructure. The N-terminal helix (αA) is positioned at the top of the ß-7

sheet. 8

The glycine-rich motif-I in PmtA (58ELGPGTG64) forms a tight loop between β-strand 9

1 and the following helix (Fig. 8B). In most MTases this so called G-loop was shown 10

to be crucial in positioning the adenine ring of SAM in its correct conformation to 11

ensure close contact with the main chain of the protein framework (10). At least one 12

of the conserved glycines in the "GxGxG" loop (motif I) was shown to contact the 13

carboxypropyl moiety or amino group of SAM (9). Thus, G60 and/or G62 in PmtA 14

might allow for hydrogen bonding of the main chain atoms to the carboxyl and amino 15

group of SAM (Fig. 8C). In analogy to various MTases of known structure and 16

function (7, 27), we speculate that E58 of motif-I coordinates SAM through a water 17

molecule. SAM-MTases are postulated to perform a nucleophilic catalysis or SN2 18

reaction (10, 27, 34, 56). The water molecule coordinated by the acidic residues 19

could either serve as a nucleophile or could aid the displacement of the bond 20

between the sulfonium ion and the methylgroup (27). 21

Motif II containing the crucial acidic amino acid E84 is found at the end of strand 2 22

(Fig. 8B). E84 is predicted to contact the ribosyl hydroxyl groups and/or adenine 23

base of SAM (Fig. 8C) like in other SAM-Mtases (7, 10, 27). 24

Although the classic SAM-binding motifs (I-III) are missing in eukaryotic Pmt 25

enzymes, two partial consensus SAM-binding motifs were identified recently in the 26


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phopholipid N-methyltransferase PEMT from mice. Like in A. tumefaciens PmtA, two 1

G and E residues (G98, G100, E180, and E181) were found to be essential for SAM 2

binding (46). 3

Based on the common architecture of SAM-MTases (29) and the order of the SAM-4

binding motifs (Fig. 1) we suggest that PmtA is sequentially organized into SAM 5

binding, catalytic and substrate-binding regions from the N- to C-terminus. This is 6

supported by our results, which show that the SAM-binding pocket is located at the 7

N-terminal region of PmtA. The C-terminus may confer substrate selectivity as 8

shown for most other SAM-MTases (27, 30). Although the PmtA model presented 9

here should be regarded as preliminary, it provides a useful basis for the rational 10

design of further mutations in particular aiming at the identification of lipid-binding 11

residues. Moreover, the well-refined NMR spectra presented in this study provide a 12

promising foundation for more detailed insights into the structural characteristics of a 13

bacterial phospolipid N-methyltransferase. 14 on June 12, 2020 by guesthttp://jb.asm

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ACKNOWLEDGEMENTS 1

We thank Christiane Fritz for excellent technical assistance and Daniel Neu for 2

advice on CD spectroscopy. We are grateful to Martin Gartmann and Gregor 3

Barchan for excellent technical assistance with the NMR experiments, Gerd Kock for 4

help with NMR data fitting and Christian Herrmann for constructive discussions. The 5

study was funded in part by a grant from the German Research Foundation (DFG, 6

NA 240/7) to FN and a fellowship from the Promotionskolleg der Ruhr-Universität 7

Bochum to MA. 8


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REFERENCES 1

2

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properties of the phospholipid N-methyltransferase PmtA from Agrobacterium 4

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2. Aktas, M., M. Wessel, S. Hacker, S. Klüsener, J. Gleichenhagen, and F. 6

Narberhaus. 2010. Phosphatidylcholine biosynthesis and its significance in 7

bacteria interacting with eukaryotic cells. Eur J Cell Biol 89:888-94. 8

3. Basturea, G. N., and M. P. Deutscher. 2007. Substrate specificity and 9

properties of the Escherichia coli 16S rRNA methyltransferase, RsmE. Rna 10

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FIGURE LEGENDS 1

2

Fig. 1. Comparative alignment of the amino acid sequence of A. tumefaciens PmtA 3

and other bacterial phospholipid N-methyltransferases using ClustalW. Highly 4

conserved similar residues are highlighted in black, ≥ 80 % conserved similar 5

residues in darker grey and ≥ 60 % conserved similar residues are shaded in lighter 6

grey. The secondary structure of A. tumefaciens PmtA model generated by the I-7

TASSER server (40) is depicted above the primary sequence. Predicted SAM motifs 8

are indicated. Arrows point out residues that were exchanged against alanine or 9

glutamic acid. The asterisks show mutated PmtA variants which were insoluble. 10

Atum_PmtA, A. tuemfaciens PmtA (AE009001); Bjap_PmtA, 11

Bradyrhizobium japonicum PmtA (NP_767321.1); Bjap_PmtX3, 12

Bradyrhizobium japonicum PmtX3, (NP_774806.1); Mloti_PmtA, Mesorhizobium loti 13

PmtA (Mll4753); ML_mlr5374, Mesorhizobium loti ORF (Mlr5374). Smel_PmtA, 14

Sinorhizobium meliloti PmtA (Accession number AF201699). 15

16

Fig. 2. Expression and activity of point mutated PmtA derivatives after expression in 17

E. coli. (A) Detection of PmtA synthesis in E. coli crude extracts via SDS-PAGE. (B) 18

Lipid formation after expression of agrobacterial PmtA derivatives in E. coli 19

BL21(DE3). Lipids of BL21(DE3) derivatives were extracted and separated by one-20

dimensional TLC. Phospholipids were visualized with Cu2SO4 solution. L, 21

BenchMarkTM Protein Ladder; V, empty vector (pET28b); WT, wild type PmtA; PG, 22

phosphatidylglycerol; CL, cardiolipin; PE, phosphatidylethanolamine; MMPE, 23

monomethyl-PE; DMPE, dimethyl-PE; PC, phosphatidylcholine. 24

25


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Fig. 3. (A) Circular dichroism analysis of the conformational state of wt and mutated 1

forms of recombinant PmtA proteins. The spectrum of each protein with a 2

concentration of 10 µM is plotted as ellipticity [mdeg] versus wavelength. (B) 3

Midpoints of the thermal unfolding transition (Tm) were used to determine the 4

conformational stability of wt PmtA. (C) Determined Tm values of mutated PmtA 5

variants. 6

7

Fig. 4: SAM-binding and lipid-binding ability of wt and mutated PmtA derivatives. (A) 8

SAM-binding activity was analyzed with 10 µM of recombinant PmtA and 100 µM of 9

S-[methyl-14C]adenosyl-L-methionine (48.8 mCi/mmol) in the presence of 100 µM of 10

PE. One hundred percent S-[methyl-14C]adenosyl-L-methionine corresponds to 11

143757 dpm. (B) Lipid binding of SAM binding defective PmtA proteins. His6-tagged 12

PmtA variants (4 nmol) were incubated with nitrocellulose strips displaying equal 13

amounts of A. tumefaciens lipids (upper panel) or 14 nmol of PE (lower panel). 14

Bound protein was detected with anti-Penta-His HRP coupled antibody (Qiagen). 15

16

Fig. 5. Plots of SAM-binding data for wt and mutated PmtA proteins. A reaction 17

volume of 50 µl contained 10 µM of PmtA, 100 µM of PE-liposomes and 0-200 µM of 18

14C -SAM. Changes in bound 14C-SAM [dpm] were plotted against SAM 19

concentration [µM]. All data sets were fitted to the equation for one site binding (see 20

Experimental Procedures) by non-linear regression using SigmaPlot 9.0. 21

22

Fig. 6. 1D 1H-NMR-spectroscopy of A. tumefaciens wt PmtA. (A) The whole 1D 1H-23

NMR-spectrum of PmtA. The sample contained 500 µl of a 125 µM PmtA and 250 24

µM PE-liposomes in 50 mM potassium phosphate buffer, pH 8 and 5 % D2O, 25

recorded at 298 K and 600 MHz. (B) NMR-titration experiments with SAM/SAH. 26


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Changes in the low field region of the 1D 1H-NMR spectrum of PmtA with increasing 1

SAM (left) or SAH (right) concentrations. 2

3

Fig. 7. SAM- and SAH-binding affinities (KD) of PmtA determined via 1D 1H-NMR-4

titration experiments. Plots of SAM- (A) and SAH-binding (B) data. Changes in the 5

relative chemical shift area/intensity were plotted against the ratio of ligand [L] to 6

protein concentration [P]. The KD values for SAM and SAH were determined 7

according to Kannt et al. (25) and Herrmann et al. (22), respectively. 8

9

Fig. 8. PmtA homology model. (A) PmtA model was generated using the online 10

server I-TASSER (40, 54) and visualized by PyMOL (http://www.pymol.org). PmtA 11

model is shown as a ribbon diagram. The fold is characterized by a central β-sheet 12

which is formed by five parallel β-strands (5, 4, 1, 2, and 3), preceeded by the anti-13

parallel β-strand 6. Experimentally proved SAM-binding residues are shown in stick 14

representation and are highlighted. (B) A closer view into the SAM-binding pocket in 15

complex with SAM. The SAM-binding cleft is made up from the C-terminal loop 16

regions following β-strands 1 to 4. SAM is shown in stick representation in yellow. 17

(C) A closer view at the putative ligand binding site in A. tumefacines PmtA model 18

showing the proposed interactions of the residues with SAM. 19


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TABLES 1

2

Table 1. Strains and plasmids used in this study. 3

a Km, kanamycin 4

Strain or plasmid Relevant characteristic(s)a Reference/source

Bacterial strain

E. coli DH5α cloning host (19)

E. coli

BL21(DE3)

expression host (50)

Plasmids

pET28b(+) Plac-T7, Km; high-copy His-

tag expression vector

Novagen, Darmstadt,

Germany

pBO832 Wt PmtA in pET28b (1)

pBO1222 PmtAE58A in pET28b This study

pBO1228 PmtAG60A in pET28b This study

pBO1223 PmtAP61A in pET28b This study



pBO866 PmtAE84A in pET28b This study

pBO871 PmtAD106A in pET28b This study

pBO1244 PmtAD106E in pET28b This study



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Table 2. Prediction and experimental estimation of PmtA secondary structure. 1

Secondary structure content [%] Algorithma

α-helices β-sheets Loops and coils

GOR IV 38,07 12,69 49,24

PHD 38,58 17,77 43,65

Stride 37,06 10,15 52,79

CDSSTR 42,00 11,00 47,00

K2D2 39,37 11,52 49,11

a GOR IV and PHD provide secondary structure predictions based on protein 2

sequence. Stride assigns secondary structure to known structures or models. 3

CDSSTR and K2D2 allow deconvolution of protein CD-spectra for experimental 4

estimation of secondary structure content. 5


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