1

Supplementary information

___________________________________________________________

Structural basis of enzymatic benzene ring reduction

Tobias Weinert1†, Simona G. Huwiler2†, Johannes W. Kung2, Sina Weidenweber1, Petra Hellwig3, Hans-Joachim Stärk4, Till Biskup5, Stefan Weber5, Julien J. H. Cotelesage6,7, Graham N. George6, Ulrich Ermler1* & Matthias Boll2*

__________________________________________________________ 1Max Planck Institute of Biophysics, Max-von-Laue-Straße 3, 60438 Frankfurt/Main, Germany. 2Microbiology, Faculty of Biology, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany 3Laboratoire de Bioélectrochimie et Spectroscopie, UMR 7140, Chimie de la Matière Complexe, Université de Strasbourg-CNRS , Strasbourg 67000, France. 4Department of Analytical Chemistry, Helmholtz Centre for Environmental Research UFZ, 04318 Leipzig, Germany. 5Institute of Physical Chemistry, University of Freiburg, Albertstr. 21, 79104 Freiburg, Germany. 6Department of Geological Sciences, University of Saskatchewan, 114 Science Place Saskatoon SK S7N 5E2, Canada 7Canadian Light Source, 101 Perimeter Road, Saskatoon, SK, S7N 0X4, Canada

*Correspondence should be addressed to ulrich.ermler@biophys.mpg.de or to matthias.boll@biologie.uni-freiburg.de †These authors contributed equally to the work

Supplementary Results

-Supplementary Figures 1-8 (page 2-9)

-Supplementary Tables 1-4 (page 10-13)

-Supplementary References (page 14)

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Supplementary Results

240 270 310 332 | | | | Geobacter metallireducens GS-15 WP_004514579.1 MPILAGLGSPQEMKVHDEKWHTENFNWGNAR... ...ISMEGLPTYMMKCFTKLTYTMAA Geobacter daltonii WP_012645313.1 MPILAGLGSPQEMKVHDEKWHTENFNWGNAR... ...ISMEGLPTYMMKCFTKLTYTMAA Geobacter sp. M21 WP_015838067.1 MPILAGLGSPQEMKVHDEKWHTENFNWGNAR... ...ISMEGLPTYMMKCFTKLTYTMAA Geobacter bemidjiensis WP_012529875.1 MPILAGLGSPQEMKVHDEKWHTENFNWGNAR... ...ISMEGLPTYMMKCFTKLTYTMAA Geobacter bremensis WP_026839972.1 MPILAGLGSPQEMKVHDEKWHTENFNWGNAR... ...ISMEGLPTYMMKCFTKLTYTMAA Desulfobacula toluolica WP_014955906.1 MPILAGLGSPQEMKVHDEKWHTENFMWGNSR... ...LSLPGLPTYMMKCFTKLTYTMGA Desulfobacula sp. TS WP_031448821.1 MPILAGLGSPQEMKVHDEKWHTENFMWGNSR... ...LSLPGLPTYMMKCFTKLTYTMGA Desulfuromonas sp. TF WP_027715408.1 MPILAGLGSPQEMKVHDEKWHTENFMWGNSR... ...ISMPGMPTYMMKCFTKLTYTMGA Desulfotignum balticum WP_024335814.1 MTILQGLGSPQEMKVHDEKWHTENFMWGNSR... ...ISMPGVPTYMMKCFTKLTYTMAA Desulfotignum phosphitoxidans WP_006965649.1 MTILQGLGSPQEMKVHDEKWHTENFMWGNSR... ...ISMPGVPTYMMKCFTKLTYTMAA Desulfomonile tiedjei WP_014809765.1 MPILAGLGSPQEMKVHDEKWHTENFMWGNSR... ...ISPPGLPTYMMKCFSKLTYTMAA Geopsychrobacter electrodiphilus WP_020678303.1 MPILAGLGSPQEMKVHDEKWHTENFMWGNSR... ...ISMPGLPTYMMKCFTKLTYTMGA delta proteobacterium PSCGC 5296 WP_027983826.1 IPILAILGSPQEMAIHDEKWHTENFSWGNAR... ...ISMPGLPTYMMKCFTKLTYTMAA Desulfococcus multivorans WP_020876803.1 MPILAGLGSPQEMAIHDEKWHTENFMWGNSR... ...ISMPGHPTYMMKCFSKLTYTMAA Desulfatiglans anilini WP_028320112.1 MTILKGLGSPQEMAVHDEKWHTENFAWGNAR... ...VSVPGLSTYMMKCFSKLTYTMAA delta proteobacterium NaphS2 WP_006422256.1 MAILTGLGSPQEMKVHDEKWHTENFMWGNSR... ...ITPPGKPTYMMKCFTKLTYTMAA Desulfatirhabdium butyrativora WP_028324510.1 MPILAGLGSPQEMKVHDEKWHTENFMWGNSR... ...ISPPGHPTYMMKCFSKLTYTMAA Deferrisoma camini WP_025324318.1 MPILAGLGSPQEMAIHDEKWHTENFMWGNSR... ...LSLPGTPTYMMKCFSKLTYTMAA Desulfospira joergensenii WP_022665735.1 TPILAVLGSPQEMAIHDEKWHTENFCWGNAR... ...LSLPGMPTYMMKCFTKLTYTMGA

Supplementary Figure 1 | Sequence alignment of conserved amino acids involved in W and Zn ligation. An

alignment of BamB of Geobacter metallireducens GS-15 (WP_004514579.1) with other BamB homologs up to a

sequence identity of 78% (query cover > 90%) using standard protein BLAST (NCBI, against non-redundant protein

sequences) is shown. Two homologous regions are shown with conserved zinc-binding motif/proton network

(Glu251, His255, Glu257 & His260, all on grey background) and active site Cys322 (white on black background).

Amino acid numbering is the same for all homologs. The conserved amino acids shown are all missing in

aldehyde:ferredoxin oxidoreductases. ClustalW Multiple alignment (1000 bootstraps)1 was used in program BioEdit

(version 7.1.7)2.

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Supplementary Figure 2 | Structural hints for the identification of the sixth non-proteinogenic ligand. The 2Fobs-

Fcalc electron density was drawn at four contour levels. Single C, N and O atoms are not visible at 6-7 σ but S and W.

Therefore an oxo or hydroxyl group can be excluded. A diatomic species CN or CO cannot be completely excluded

because of the small distance between C and N (O) and the vicinity of the electron-rich W.

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Supplementary Figure 3 | FT-IR spectrum of BamBC. The wavenumber region where potential vibrational signals

from diatomic ligands would have been expected is shown. The signals at 1646 and 1540 cm-1 are assigned to the

amide I and II signals, respectively. The two signals shown in the magnified inset are too broad to be assigned to a

diatomic ligand.

Wavenumber / cm-1140015001600170018001900200021002200

0.00

0.05

0.10

0.15

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Supplementary Figure 4 | Zn K-edge EXAFS of BamBC. The blue lines show experimental data and red lines the

best fit corresponding to that with two histidine ligands detailed in Supplementary Table 4. The transform peak at

~3.9 Å corresponds to the outer C4 and Nπ atoms of two histidine imidazole ligands, and the intensity of this peak is

diagnostic of the number of such ligands to the metal. Fits were done using a full multiple scattering approach but

approximating the histidine imidazole as a rigid body, and assuming that the two glutamate residues are equivalent.

These assumptions are made necessary because of the limited k-range which is due to the proximity of the W LIII

edge.

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Supplementary Figure 5 | Binding of monoenoyl-CoA (green), dienoyl-CoA (grey) and benzoyl-CoA (yellow) in

BamBCCoA structures. The structures with the three CoA esters are superimposed. The electron density for

monoenoyl-CoA is given at a contour level of 1.5 σ. In the four BamBC units of the asymmetric unit of the

Bam(BC)2-benzoyl-CoA complex, only two of the four benzoyl-CoA molecules are significantly occupied. Compared

to monoenoyl-CoA and dienoyl-CoA the six-membered ring of benzoyl-CoA is only weakly visible perhaps due to a

partial hydrolysis of the thioester bond.

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Supplementary Figure 6 | Superposition of the BamBCisol and BamBCCoA state in stereo. The BamBCisol state

(carbon in yellow) based on crystals grown from the purified Bam(BC)2 complex and the BamBCCoA state (carbon in

grey) based on the Bam(BC)2-monoenoyl-CoA complex are shown around the active site.

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Zinc chloride (µM)

1/A

ctiv

ity (U

-1m

g)

-20 -15 -10 -5 0 5 10 15 20 25 300.0

0.2

0.4

0.6

0.8

1.0

Supplementary Figure 7 | Inhibition of BamBC from G. metallireducens by Zn2+. Dixon plot analysis of BamBC

activity determined in the reverse reaction at different dienoyl-CoA concentrations revealed a Ki = 6.7 ± 0.5 µM

( 20 µM, 40 µM, 80 µM dienoyl-CoA). Linear regression lines were fitted to all data points including

inhibition at 50 µM and 100 µM zinc chloride (not shown here). Data represent mean values ± standard deviation of

samples measured at duplicates or triplicates. Data points without standard deviation were measured once (except for

40 µM dienoyl-CoA and 3.1 µM zinc chloride where the standard deviation is too small to be seen in the figure).

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Supplementary Figure 8 | Proposed mechanism of Birch reduction-like mechanism of benzoyl-CoA reductase

catalysis. Red arrows indicate electron transfer, blue arrows protonation events with the proton-donating amino acids

indicated as determined by the BamBCCoA structures.

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Supplementary Tables

Supplementary Table 1 | Crystal forms of Bam(BC)2

Crystal Crystallization Cryo protection / soaking

BamBCCoA (monoenoyl-CoA)

8% (w/v) PEG 4000 0.2 M LiCl 2 mM DTT 0.2 M Tris pH 8.5 2 µl : 2 µl drop ratio 4°C

8% (w/v) PEG 4000 30% (w/v) PEG 400 0.2 M LiCl 0.2 M MES pH 6.5 10 mM monoenoyl-CoA soaking 6 h at 4°C

BamBCCoA (dienoyl-CoA)

4.2 % (w/v) PEG 4000 0.2 M LiCl 2 mM DTT 1 mM ZnSO4 0.2 M Tris pH 8.5 2 µl : 2 µl drop ratio 4°C

5% (w/v) PEG 4000 30% (w/v) PEG 400 0.2 M LiCl 0.1 M MES pH 6.5 1 mM ZnSO4 5 mM dienoyl-CoA soaking 18h at 4°C

BamBCCoA (benzoyl-CoA)

7 % (w/v) PEG 4000 0.2 M LiCl 0.1 M Tris pH 8.5 1 µl : 1 µl drop ratio 18°C

5% (w/v) PEG 4000 30% (w/v) PEG 400 0.2 M LiCl 0.1 M MES pH 6.5 10 mM benzoyl-CoA soaking 4h at 4 °C

BamBCisol (as isolated)

6 % (w/v) PEG 4000 0.2 M LiCl 0.2 M Tris pH 8.5 2 mM DTT 1 µl : 2 µl drop ratio 4°C

6 % (w/v) PEG 4000 30% (w/v) PEG 400 0.2 M LiCl 0.1 M Tris pH 7.5

BamBCisol (Zn-absorption edge)

3.8 % (w/v) PEG 4000 0.2 M LiCl 2 mM DTT 1 mM ZnSO4 0.2 M Tris pH 8.5 2 µl : 2 µl drop ratio 4°C

5% (w/v) PEG 4000 30% (w/v) PEG 400 0.2 M LiCl 0.2 M Tris pH 8.5 1 mM ZnSO4 soaking 4h at 4°C

BamBCisol (W-absorption edge)

8% (w/v) PEG 4000 0.2 M LiCl 2 mM DTT 0.1 M Tris pH 8.5 1 µl : 1 µl drop ratio 18°C

8% (w/v) PEG 4000 30% PEG 400 0.2 M LiCl 0.1 M Tris pH 7.5 no soaking

The three BamBCCoA and BamBCisol structures were almost identical, respectively.

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Supplementary Table 2 | Data collection and refinement statistics

BamBCCoA

(monoenoyl-CoA)

BamBCCoA

(dienoyl-CoA)

BamBCCoA

(benzoyl-CoA)

BamBCisol

(Zn edge)

BamBCisol BamBCisol

(W edge)

Data collection

Wavelength (Å) 1.250 0.999 1.000 1.282 1.000 1.214

Space group P21 P21 P21 P21 P21 P21

Cell dimensions

a, b, c (Å)

125.8, 116.3, 144.1

125.3, 116.3, 144.2

125.0, 116.1, 142.5

124.3, 117.5, 143.1

125.2, 116.8, 143.6

124.5, 116.3, 142.5

α, β, γ (°) 90, 110.4, 90 90, 110.5, 90 90, 111.4, 90 90, 111.0, 90 90, 110.4, 90 90, 110.7, 90

Resolution (Å) 50.0-1.9

(2.18-1.9)*

50.0-2.4

(2.5-2.4) *

50.0-2.5

(2.6-2.5)

50.0-3.0

(3.1-3.0)

50-2.11

(2.16-2.11)

50.0-2.7

(2.8-2.7)

Rsym 8.0 (57.9) 10.4 (69.8) 8.1 (68.8) 10.6 (72.4) 20.1 (1228.3) 8.5 (41.2)

I/σI 7.3 (2.2) 8.5 (2.2) 10.4 (2.5) 6.9 (1.5) 4.26 (0.15) 6.5 (1.9)

Completeness (%) 98.7 (99.4) 99.2 (96.2) 98.9 (98.7) 94.6 (82.1) 95.8 (68.1) 96.5 (97.1)

Redundancy 2.8 (2.9) 4.1 (3.6) 3.8 (3.9) 2.6 (2.3) 4.5 (3.2) 2.1 (2.2)

Refinement

Resolution (Å) 48.2-1.9 46.9-2.3 46.7-2.8 47.7-2.9 49.5-2.1

No. reflections 293278 165056 148322 157594 208029

Rwork / Rfree 23.0/20.7 22.8/20.4 27.2/22.3 27.8/24.4 23.3/25.8

No. atoms

Protein 25865 25874 25767 25833 25838

Ligand/ion 655 612 442 336 336

Water 1137 475 0 0 208

B-factors (Å2)

Protein 58.2 65.6 74.1 97.3 102.5

Ligand/ion 55 65.2 62.4 69.7 79.6

Water 47.2 47.6 - - 71.9

R.m.s deviations

Bond lengths (Å) 0.01 0.004 0.005 0.005 0.009

Bond angles (º) 1.09 0.796 0.848 0.670 0.920

Ramachandran

favored/ allowed/

disallowed (%)

7.7/ 2.3/ 0.0

96.7/ 3.1/ 0.2

94.5/ 4.7/ 0.8

93.8/ 5.7/ 0.5

95.5/ 3.9/ 0.6

*Highest resolution shell is shown in parenthesis.

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Supplementary Table 3 | W LIII EXAFS curve fitting results* for BamBCisol Model Ligand N R (Å) σ2 (Å2) ∆E0 F

a W—S 5 2.422(1) 0.0021(1) -11.4(4) 0.179

W—C 1 2.014(4) 0.0020(4)

W···CN 1 3.204(4) 0.0020†

b W—S 4 2.421(1) 0.0014(1) -11.7(4) 0.209

W—C 1 2.011(4) 0.0020(4)

W···CN 1 3.201(4) 0.0020†

c W—S 6 2.425(1) 0.0032(1) -11.0(4) 0.215

W—C 1 2.021(4) 0.0033(4)

W···CN 1 3.211(4) 0.0033†

d W—S 5 2.423(1) 0.0015(1) -11.0(4) 0.233

W—Cl 1 2.619(16) 0.0070(18)

*Coordination numbers (N), interatomic distances (R), Debye-Waller factors (σ2), and threshold energy shift ΔE0 (eV).

The fit error F is given by normalized F-value ( ) ( )( ) ( ) −= 2626exptexptcalc kkkkkF χχχ , with the summation being

over data points included in the fit. Values in parentheses are the estimated standard deviations obtained from the

diagonal elements of the covariance matrix; these are precisions and are distinct from the accuracies which are

expected to be larger (ca ± 0.02 Å for R, and ± 20 % for N and σ2), and that relative accuracies (e.g. comparing two

different W–S bond-lengths) will be more similar to the precisions. The amplitude scale factor, otherwise known as

the many-body amplitude reduction factor, or S02, was defined by fitting data from a number of model compound

species as 1.0. In all cases the k-range of the data fitted was from 1.0 to 14 Å-1. All σ2 values are within chemically

reasonable ranges. Models a-c show the effect of varying the W–S coordination number, and model d shows the effect

of including chloride, a possible alternative sixth ligand, which (apart from having a poorer F) lacks the peaks in the

Fourier transform at 2.0 and 3.2 Å that are apparent in Fig. 3b. †The strength of the C≡N bond means that this can be treated as a rigid group in EXAFS analysis, enabling the

simplifying assumption that the σ2 value for N is identical to that for C. The multiple scattering EXAFS calculations

included both three and four leg multiple scattering paths.

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Supplementary Table 4 | Zn K EXAFS curve fitting results* for BamBCisol Model Ligand N R (Å) σ2 (Å2) ∆E0 F

a Zn—O(Glu) 3 2.025(18) 0.0092(29) -6.3(5) 0.415

Zn—N(His) 1 1.979(26) 0.0037(11)

b Zn—O(Glu) 2 2.042(18) 0.0075(18) -6.2(5) 0.394

Zn—N(His) 2 1.975(26) 0.0036(10)

c Zn—O(Glu) 1 2.074(18) 0.0131(32) -6.4(5) 0.474

Zn—N(His) 3 1.984(19) 0.0036(10)

*See Supplementary Table 3 for symbols and other details. Model b corresponds to the fit shown in

SupplementaryFigure 4.

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Supplementary References 1. Thompson, D. J., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of

progressive multiple sequence alignment through sequence weighting, position-specific gap

penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680 (1994).

2. Hall, T. A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for

Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95-98 (1999).

Nature Chemical Biology: doi:10.1038/nchembio.1849