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1 Supplementary Information Using the pimeloyl-coa synthetase adenylation fold to synthesise fatty acid thioesters Menglu Wang 1a ; Lucile Moynié 2a, Peter J. Harrison 1, Van Kelly 1, Andrew Piper 1, James H. Naismith 2, 3* and Dominic J. Campopiano 1* 1 EastChem School of Chemistry, David Brewster Road, University of Edinburgh, Edinburgh, EH9 3FJ. 2 Biomedical Sciences Research complex, University of St. Andrews, St Andrews, KY16 9ST 3 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital of Sichuan University and Collaborative Innovation Center of Biotherapy and Cancer, Chengdu , China 1
2 Supplementary Results (a) MSYYHHHHHH DYDIPTTENL YFQGAMEEET FYSVRMRASM NGSHEDGGKH 27 ISGGERLIPF HEMKHTVNAL LEKGLSHSRG KPDFMQIQFE EVHESIKTIQ 77 PLPVHTNEVS CPEEGQKLAR LLLEKEGVSR DVIEKAYEQI PEWSDVRGAV 127 LFDIHTGKRM DQTKEKGVRV SRMDWPDANF EKWALHSHVP AHSRIKEALA 177 LASKVSRHPA VVAELCWSDD PDYITGYVAG KKMGYQRITA MKEYGTEEGC 227 RVFFIDGSND VNTYIHDLEK QPILIEWEED HDS 259 Supplementary Figure 1a. Amino acid sequence of B. subtilis 6His BioW. The sequence of the full length clone and recombinant BioW after TEV cleavage to remove the His tag is shown. The BioW numbering begins at Ala1 and ends with Ser259. Residues in the active site are in bold (Tyr199, Tyr211, Arg213 and Arg227) and the His tag and TEV cleavage site in blue and red respectively. (b) Supplementary Figure 1b. 15% SDS-PAGE gel showing the purity of purified BioW enzyme. M: LMW Marker, Lane 1: E. coli cell pellet, Lane 2: Cell lysate, Lane 3: Ni resin flow through, Lane 4: Ni resin wash, Lane 5: Ni resin elution, Lane 6: Pre-TEV protease cleavage, Lane 7: Second Ni resin flow through, Lane 8: Second Ni resin elution, Lane 9-12: Superdex 200 fractions A band at ~30 kda corresponds to the BioW molecular weight. 2
3 (c) Absorbance (280 nm) (mau) Retention Volume (ml) BioW V e = 73.4 ml Supplementary Figure 1c. Chromatogram of purified BioW. The purified BioW was applied to a calibrated Superdex S200HR gel filtration column (A280nm). BioW elutes at 73.4 ml which corresponds to a homodimer. 3
4 (a) (b) 4
5 (c) (d) 5
6 (e) Supplementary Figure. 2. Activity of recombinant B. subtilis BioW. (a) ATP (retention time 3.1 mins), ADP (4.0 mins) and AMP (4.2 mins) standards (Sigma) co-injected onto the C18 RP HPLC column. (b) Co-injection of ATP with Coenzyme A from Sigma (retention time 13.3 mins). (c) Synthetic pimeloyl-coa (retention time mins, see Methods). (d) ESI FT-ICR MS analysis of synthetic pimeloyl-coa (m/z range = ). Inset shows the ion with mass = Da consistent with [M+H] +. Predicted mass = Da (C 28 H 46 N 7 O 19 P 3 S). The red circles represent the isotopic distribution predicted from the chemical formula. (e) BioW-catalysed pimeloyl-coa synthesis. The incubation described in the Methods was analysed by HPLC and we observed peaks corresponding to pimeloyl-coa at 16.2 mins and AMP (4.2 mins). The inset show the ESI FT-ICR MS analysis of the peak at 16.2 mins which agrees well with the synthetic standard shown in (c). 6
7 Supplementary Figure 3. Basis of the coupled BioW activity assay. Pimelic is converted to pimeloyl-coa by BioW and the pyrophosphate (PPi) released is hydrolysed to two molecules of phosphate (Pi) by pyrophosphatase (PPase). Purine nucleoside phosphorylase (PNP) catalyses the reaction between the released P i and MESG to give the product 7-methyl- 6-thioguanine (which absorbs at 360 nm, ε 360 = 11,000 M -1 cm -1 ) and ribose phosphate. 7
8 (a) (b) (c) Supplementary Figure 4. Kinetic analysis of WT BioW using the coupled assay. Substrates (a) pimelic acid and (b) ATP. (c) Determination of the kinetic data for CoASH 8
9 used the 230nm UV assay. Data was fit using Michaelis-Menten model on GraphPad. Data represent mean values ± s.d. from multiple experiments. 9
10 10
11 11
12 h) 12
13 Supplementary Figure 5. Structures of BioW. (a) Binding sites of pimeloyl-adenylate and PPi. For more clarity, only the polar interactions have been represented and the figure has been split in two views, one for the AMP moiety (left) and the other for the pimeloyl group (right). Carbon atoms of the pimeloyl-adenylate are in cyan. Hydrogen bonds are shown as black dashes lines. Oxygen atoms are in red, nitrogen in blue, phosphate in orange and Mg 2+ ions are shown as green spheres. (b) Binding sites of AMP-PNP and pimelic acid. The colour scheme is the same than a) but the carbon atoms of AMP-PNP and pimelic acid are in yellow. (c) Fo-Fc electron density omit map at 3σ around AMP-PNP and pimelic acid. (d) Ligplot diagram of pimeloyl-adenylate/ppi. Covalent bonds of the ligands and protein residues are in purple and brown sticks, respectively. Hydrogen bonds are represented by green dashed line and hydrophobic contacts are shown as red semi-circles with radiating spokes. Figure prepared with Ligplot 1. (e) Ligplot diagram of AMP-PNP/pimelic acid. (f) Ligplot diagram of the adenosine 3', 5'-diphosphate group of CoA bound to BioW. (g) Fo-Fc electron density omit map at 3σ around the ordered portion of CoA. (h) Two views of the electrostatic surface of the BioW with the ordered portion of CoA shown as spheres. There are two possible routes to the active site denoted with dashed circle. Positively charged regions are shown in blue and negatively regions are in red. Oxygen atoms are in red, nitrogen in blue, phosphate in pink. 13
14 Supplementary Figure 6. Sequence alignment of bacterial BioW. The sequences used are from Bacillus subtilis (UNIPROT code: P53559, BioW_BACSU), Aquifex aeolicus (O67575, BioW_AQUAE), Lysinibacillus sphaericus (P22822, (IOW_LYSSH), and Staphylococcus aureus (P67549, BioW_STAAM). The residues targetted for engineering the fatty acid specificity (Y199, R211, R213, R227) are marked with blue triangles. Figure produced using ESPript
15 Supplementary Figure 7. The BioW Y211F mutant displays broadened fatty acid specificity. The turnover (k cat, s -1 ) of each BioW enzyme, WT and mutants Y199F, Y211F and R213A (3.0 µm) in the presence of different carboxylic acids (1.5 mm), ATP (1.0 mm) and CoASH (1.0 mm). Data represent mean values ± s.d. from multiple experiments. 15
16 Supplementary Figure 8. Kinetic analysis of the BioW R11A and R13A mutants with pimelic acid as a substrate. The data was obtained using the PPi assay and the WT activity is included for comparison. Data represent mean values ± s.d. from multiple experiments. 16
17 Supplementary Figure 9. Kinetic analysis of BioW Y211F. We used BioW Y211F (2.5 µm) and different concentrations of heptanoic acid (top panel) and ATP (lower panel) as substrates. The data were obtained using the PPi assay and the kinetic parameters for each substrate were determined. The data was fitted using the Michaelis-Menten model on GraphPad; heptanoic (k cat = s -1 ± , K m = ± 79.7) and ATP (k cat = ± s -1, K m = ± 81.5 µm). Data represent mean values ± s.d. from multiple experiments. 17
18 (a) Pimeloyl-CoA * WT Time (mins) * Y211F Base Peak Base Peak Intensity Time (mins) m/z (b) 7-Octenoyl- WT Base Peak Intensity Base Peak Intensity Time * Y211F Time (mins) m/z 18
19 (c) 7-Phenylheptanoyl-CoA WT SCoA Base Peak O 7-Phenylheptanoyl-CoA Time (mins) Y211F Base Peak * m/z Time (mins) (d) 6-Methylheptanoyl-CoA WT Base Peak Time (mins) * Y211F Base Peak Time (mins) m/z 19
20 (e) 7-Bromoheptanoyl-CoA WT Base Peak Intensity Base Peak Intensity Time (mins) * Y211F (f) 7-Aminoheptanoyl-CoA Time (mins) m/z WT Base Peak Intensity Time (mins) Y211F Expected [M+H] not observed Base Peak Time (mins) Supplementary Figure 10. Mass spectrometry analysis of acyl-coa products. Base peak intensity chromatograms are presented for LCMS analyses of reactions using six substrates; (a) pimelic acid, (b) 7-octenoic acid, (c) 7-phenyl-heptanoic acid, (d) 6-methylheptanoic acid, (e) 7-bromo-heptanoic acid and (f) 7-aminoheptanoic acid for both BioW WT and Y211F mutant enzymes. Chromatographic peaks corresponding to the expected acyl-coa thioester 20
21 product masses are annotated (*) and the mass spectrum of the [M+H] + ion is presented, if observed. The observed monoisotopic mass annotation was extracted from centroided data and all masses were observed within 3ppm from the expected mass. The acyl-coa thioester product was produced by the BioW Y211F mutant for all substrates tested, apart from F) 7- aminoheptanoic acid. 21
22 Supplementary Table 1: Kinetic parameters of BioW. The pimelic acid and ATP data were determined using the PPi coupled assay and the CoASH data using thioester bond formation at 230nm assay (*). Data represent mean values ± s.d. from multiple experiments. Pimelic acid ATP CoASH* K m (µm) 70.5 ± ± ± 25.4 k cat (s -1 ) 0.48 ± ± ± 0.05 k cat / K m (M -1 s -1 ) 8, , ,
23 Supplementary Table 2. Chain length specificity of WT BioW. Turnover values (k cat ) of the dicarboxylic and monocarboxylic acid substrates of different chain lengths. The values were determined using the PPi assay (see Methods). Common name Carbon Chain Length Turnover (s -1 ) Glutaric acid Adipic acid Pimelic acid Suberic acid Azelaic acid Heptanoic acid Octanoic acid
24 Supplementary Table 3: Crystallographic data and refinement statistics K 2 PtCl 4 Pimeloyladenylate/PPi complex (5fm0) AMP- PNP/pimelic acid complex (5flg) Pimeloyladenylate/PPi complex (5fll) Pimelic acid/coash complex (5g1f) Data collection Space group P P P P Cell dimensions a, b, c (Å) , 77.90, , 78.11, , 78.36, α, β, γ ( ) 90.0, 90.0, , 90.0, , 90.0, , 90.0, 90.0 Resolution (Å) ( )* ( ) ( ) ( ) R sym or R merge (0.681) (0.552) (0.419) (0.757) I / σi 23.3 (3.3) 17.3 (2.7) 21.4 (3.36) 18.8 (2.0) Completeness (%) 99.9 (99.9) 99.2 (92.2) 99.5 (94.2) 99.8 (99.7) Redundancy 15.2 (10.5) 6.4 (4.1) 4.2 (2.9) 6.4 (5.1) Refinement Resolution (Å) No. reflections R work / R free 0.200/ / / / No. atoms Protein Ligand/ion Water B-factors Protein Ligand/ion Water R.m.s deviations Bond lengths (Å) Bond angles ( ) Each dataset was collected from a single crystal. *Values in parentheses are for highest-resolution shell. 24
25 Supplementary References 1 Wallace, A. C., Laskowski, R. A. & Thornton, J. M. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein engineering 8, (1995). 2 Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic acids research 42, W , doi: /nar/gku316 (2014). 25
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