Supplementary Material

Table of Contents:

Synthesis of S2d

Figure S1. Comparison of previously proposed N-terminal sequences.

Figure S2. ORF12 kinetics.

Figure S3. Assay for DD-carboxypeptidase activity.

Figure S4. Phylogenetic relationships between ORF12, EstB, -lactamases and PBPs.

Figure S5. Bocillin assay.

Figure S6. Sequence alignment with close homologues of ORF12.

Figure S7. Sequence alignment with close homologues of ORF12 including LpqF subfamily.

Figure S8. Phylogenetic diagram showing the evolutionary relationship of ORF12.

Figure S9. Possible epimerisation role of ORF12.

Table S1. Cephalosporin substrates and non-substrates for ORF12.

Table S2. Comparison of catalytic residues at the active site of PBP/β-lactamase-fold

enzymes.

References

Synthesis of S2d

(R)-2-((2-benzamidopropanoyl)thio)acetic acid

To a solution of (R)-2-benzamidopropanoic acid3 (0.1 g, 0.52 mmol) and triethylamine (0.1

ml, 0.68 mmol) in dry THF (5 ml), ethyl chloroformate (50 μl, 0.57 mmol) was added drop

wise over 1 min at 0 °C. The mixture was stirred for 40 min and a solution of thioglycolic

acid (0.11 ml, 1.56 mmol) in THF and triethylamine (0.25 ml, 1.56 mmol) was added

dropwise over 2 min at the same temperature. The solution was left to warm to r. t. and

stirred at 323 K for 3 hours. The solvent was removed under reduced pressure. 3 ml of 1M

HCl were added in the crude and were left to stir for 5 min before it was washed with EtOAc.

The organic phase was dried MgSO4 and concentrated in-vacuo and the crude was purified by

HPLC. Trt = 9 min. IR (neat) ν/cm-1

3345 (NH), 1728 (CH3OC=O), 1667(RHNC=O); 1H

NMR (500 MHz; CDCl3) δ 8.29-8.24 (1H, m, Ar CH), 7.59-7.53 (

1H, m, NH), 7.49-7.42 (

1H,

m, Ar CH), 6.85 (1H, s, Ar CH), 4.97 (

2H, d, J = 5.5, CH2), 3.29-3.19 (

3H, s, CH3),

13C NMR

(125MHz, CDCl3) δ 170.0 (C=O), 163.8 (C=O), 149.5, 147.7, 140.0, 124.3, 123.7, 52.4

(CH3), 41.25 (CH2); MS (ESI+) m/z 273 ([M+H]+, 100%); HRMS calcd. for C9H9BrN2NaO3

[M+Na]+ 294.9689; Found 294.9689.

Figure S1. Alternative N-terminal sequences (a) from Jensen et al. 5 and used in this work;

(b) from Mellado et al. 7; (c) from Li et al.

11.

(a) MAEGRQGAMMKKADSVPTPAEAALAAQTALAADDSPMGDAARWAMGLLTSSGLPRPEDVA

(b) MGDAARWAMGLLTSSGLPRPEDVA

(c) MAEGRQGAMMKKADSVPTPAEAALAAQTALAADDSPMGDAARWAMGLLTSSGLPRPEDVA

Figure S2. ORF12 kinetics. (a) Michaelis-Menten plot of kinetic data obtained for wild type

ORF12 with cephalosporin C as determined by HPLC with (b) residual plot, (c) Lineweaver-

Burk plot with (d) residuals, ((e) Hanes-Woolf and (f) NMR monitoring of ORF12 activity in

real time; signals for cephalosporin C and acetate.

.

Figure S3. Assay of Streptomyces clavuligerus ORF12 for D,D-carboxypeptidase activity with peptidoglycan precursor pentapeptides. Assays were performed in a final volume of

0.2 ml, containing 50 mM HEPES, 10 mM MgCl2, 50 mM KCl, 0.1 mM amplex Red, 0.27

mg.ml-1

horse radish peroxidase, 0.38 mg·ml-1

D-amino acid oxidase with ORF12 added to

0.11 mg·ml-1

at 1. Absorbance at 555 nm at 310 K was followed and the assay was

supplemented in (a) with 3.2 mM UDP-MurNAc pentapeptide (lys) at 2, a further 0.44

mg.ml-1

ORF12 at 3, 3.2 mM UDP-MurNAc pentapeptide (DAP) at 4 and 82 g.ml-1

E. coli

DacB at 5. The final addition (5) was a positive D,D-carboxypeptidase control (DacB) to test

the functionality of the assay or, in (b) with 3.2 mM UDP-MurNAc pentapeptide (DAP) at 2

and 82 g·ml-1

E. coli DacB at 3. The final addition (3) was a positive D,D-carboxypeptidase

control (82 g·ml-1

E. coli DacB) to test the functionality of the assay.

Figure S4. Phylogenetic tree adapted from both Koch 15

and Massova and Mobashery 17

showing the relationship of ORF12 with PBPs, -lactamases, and the esterase EstB.

Figure S5. Bocillin assay. Comparative reactivity of Streptomyces clavuligerus ORF12, E.

coli DacB, PBP2b and PBP2x from Streptococcus pneumoniae with fluorescent bocillin:

PBPs (20 µg) were incubated in 20 µl in 50 mM HEPES, 10 mM MgCl2, pH 7.6, for 30 min

(Room Temp.) with or without 0.1 mM ampicillin. Samples were then supplemented with

bocillin at a 5:1 molar ratio of protein to bocillin and incubated for a further 1 hr. Samples

were then subjected to SDS PAGE (10% acrylamide). a. Polyacrylamide gel analysed for

bocillin-labelled PBP protein fluorescence using a Syngene GelSnap G Box Gel Doc with a

blue light converter and filter for detecting fluorescence emission at 520 nm. b. Gel in Figure

S5 (a) subsequently stained for total protein with Coomassie Blue.

Figure S6. Sequence alignment of the two closest homologues to ORF12 identified in the

NCBI database. StrepC (ORF12), gi|254387594, ref|ZP_05002833.1, Streptomyces

clavuligerus ATCC 27064; StrepF, gi|320007159, gb|ADW02009.1, Streptomyces

flavogriseus ATCC 33331; SaccV, gi|257057305, ref|YP_003135137.1, Saccharomonospora

viridis DSM 43017.

Figure S7. Sequence alignment of ORF12 with close homologs. Orf12, gi|254387594,

ref|ZP_05002833.1, Streptomyces clavuligerus ATCC 27064; StrepF, gi|320007159,

gb|ADW02009.1, Streptomyces flavogriseus ATCC 33331; SaccV, gi|257057305,

ref|YP_003135137.1, Saccharomonospora viridis DSM 43017; MycbSpyr1, gi|315446233,

ref|YP_004079112.1, Mycobacterium sp. Spyr1; MycbG, gi|145222026,

ref|YP_001132704.1, Mycobacterium gilvum PYR-GCK; MycbV,

gi|120406301, ref|YP_956130.1, Mycobacterium vanbaalenii PYR-1; MycbKMS,

gi|119870866, ref|YP_940818.1, Mycobacterium sp. KMS; MycbMCS, gi|108801715,

ref|YP_641912.1, Mycobacterium sp. MCS; MycbA, gi|169627656, ref|YP_001701305.1,

Mycobacterium abscessus ATCC 19977; MycbJLS, gi|126437702, ref|YP_001073393.1,

Mycobacterium sp. JLS; MycbK, gi|240172186, ref|ZP_04750845.1, Mycobacterium kansasii

ATCC 12478; MycbS, gi|118470558, ref|YP_890308.1, Mycobacterium smegmatis str. MC2

155; ClavM, gi|148271516, ref|YP_001221077.1, Clavibacter michiganensis subsp.

michiganensis NCPPB 382; SanguK, gi|269793742, ref|YP_003313197.1, Sanguibacter

keddieii DSM 10542.

Figure S8. Phylogenetic relationship of ORF12 with its closest homologues labelled by taxa.

ORF12 is marked with a red star. Background color indicates grouping into subfamilies.

Phylogenetic analysis performed using NCBI BLAST Tree View Widget (settings were Fast

Minimum Evolution, Max Sequence Difference 0.7, Distance Grishin (protein). Figure

generated using FigTree v1.3.1 (options – radial tree layout, trees:order nodes increasing,

cladogram). Note: Mycobacterium subfamily (pink) poses the “KTG” motif but the active

site Ser378

in ORF12 is replaced with an aspargine. The cyanobacterium subfamily members

(green) do not have a “KTG” motif nor a conserved Ser378

. All but the cyanobacterium

subfamily are high GC content gram(+) bacteria. gi|254387594, ref|ZP_05002833.1,

Streptomyces clavuligerus ATCC 27064; gi|320007159, gb|ADW02009.1, Streptomyces

flavogriseus ATCC 33331; gi|257057305, ref|YP_003135137.1, Saccharomonospora viridis

DSM 43017; gi|315446233, ref|YP_004079112.1, Mycobacterium sp. Spyr1; gi|145222026,

ref|YP_001132704.1, Mycobacterium gilvum PYR-GCK; gi|120406301, ref|YP_956130.1,

Mycobacterium vanbaalenii PYR-1; gi|119870866, ref|YP_940818.1, Mycobacterium sp.

KMS; gi|108801715, ref|YP_641912.1, Mycobacterium sp. MCS; gi|169627656,

ref|YP_001701305.1, Mycobacterium abscessus ATCC 19977; gi|126437702,

ref|YP_001073393.1, Mycobacterium sp. JLS; gi|240172186, ref|ZP_04750845.1,

Mycobacterium kansasii ATCC 12478; gi|118470558, ref|YP_890308.1, Mycobacterium

smegmatis str. MC2 155; gi|148271516, ref|YP_001221077.1, Clavibacter michiganensis

subsp. michiganensis NCPPB 382; gi|269793742, ref|YP_003313197.1, Sanguibacter

keddieii DSM 10542; gi|56750069, ref|YP_170770.1, Synechococcus elongatus PCC 6301;

gi|81300412, ref|YP_400620.1, Synechococcus elongatus PCC 7942; gi|257059487,

ref|YP_003137375.1, Cyanothece sp. PCC 8802; gi|254416694, ref|ZP_05030444.1,

Microcoleus chthonoplastes PCC 7420; gi|218246445, ref|YP_002371816.1, Cyanothece sp.

PCC 8801; gi|86605517, ref|YP_474280.1, Synechococcus sp. JA-3-3Ab; gi|317970457,

ref|ZP_07971847.1, Synechococcus sp. CB0205; gi|119494566, ref|ZP_01624704.1, Lyngbya

sp. PCC 8106; gi|86609350, ref|YP_478112.1, Synechococcus sp. JA-2-3B'a(2-13);

gi|227825073, ref|ZP_03989905.1, Acidaminococcus sp. D21; gi|307151215,

ref|YP_003886599.1, Cyanothece sp. PCC 7822; gi|334117135, ref|ZP_08491227.1,

Microcoleus vaginatus FGP-2;

Figure S9. Three possible mechanisms (a, b, and c) by which ORF12 may be involved in the

epimerisation process required to convert (3S,5S)-clavaminic acid to (3R,5R)-CA.

Table S1: Potential cephalosporin substrates tested with ORF12.

Compounds tested with ORF12 esterase activity

observed

enzymes. Classification based on Sauvage et al. 20

Class/Type/ Subtype Protein PDB code Serine/Lysine

(SXXK)

Serine

([S/Y]DN)

KTG

box

β-Lactamase Class A TEM-1 1M40 1 Ser

70/ Lys

73 Ser

130 Lys

234

Esterase family X ORF12 2XEP Ser173

/ Lys176

Ser234

Lys375

PBP Class C / Type-5 PBP5 1Z6F 2 Ser

44/Lys

47 Ser

110 Lys

213

PBP Class C / Type-4 R39 DD-peptidase 1W79 4 Ser49/Lys52 Ser298 Lys410

PBP Class B / Type-B1 PBP2a 1VQQ 6 Ser403/Lys406 Ser462 Lys597

PBP Class A / Type-A3 PBP1a 2C6W 8 Ser

370/Lys

373 Ser

428 Lys

557

PBP Class C /Type-7 K15 DD-peptidase 1SKF 9 Ser25/Lys38 Ser96 Lys213

β-Lactamase Class D OXA10 1E3U 10

Ser67

/ Lys70

Ser119

Lys214

β-Lactamase Class C P99 1BLS 12

Ser64

/ Lys67

Tyr150

Lys315

PBP Class C / Type-

AmpH

R61

DD-peptidase/

transpeptidase

3PTE 13

Ser62

/Lys65

Tyr159

His298

D-Aminopeptidase DAP 1EI5 14

Ser62

/Lys65

Tyr153

His287

D-Amino acid amidase DAA 2DRW 16

Ser60

/Lys63

Tyr149

His307

Esterase family VIII EstB 1CI8 18

Ser75

/ Lys78

Tyr133

Tyr181

Nylon amidase NylC 1WYC 19

Ser112

/Lys115

Ser217

Tyr215

Table S2. Catalytic residues (highlighted red) at the active site of PBP/β-lactamase-fold

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