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Using the pimeloyl-CoA synthetase adenylation fold to synthesize fatty acid thioesters

Nature Chemical Biology volume 13, pages 660–667 (2017)Cite this article

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Abstract

Biotin is an essential vitamin in plants and mammals, functioning as the carbon dioxide carrier within central lipid metabolism. Bacterial pimeloyl-CoA synthetase (BioW) acts as a highly specific substrate-selection gate, ensuring the integrity of the carbon chain in biotin synthesis. BioW catalyzes the condensation of pimelic acid (C7 dicarboxylic acid) with CoASH in an ATP-dependent manner to form pimeloyl-CoA, the first dedicated biotin building block. Multiple structures of Bacillus subtilis BioW together capture all three substrates, as well as the intermediate pimeloyl-adenylate and product pyrophosphate (PPi), indicating that the enzyme uses an internal ruler to select the correct dicarboxylic acid substrate. Both the catalytic mechanism and the surprising stability of the adenylate intermediate were rationalized through site-directed mutagenesis. Building on this understanding, BioW was engineered to synthesize high-value heptanoyl (C7) and octanoyl (C8) monocarboxylic acid-CoA and C8 dicarboxylic-CoA products, highlighting the enzyme's synthetic potential.

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Figure 1: The BioW reaction, highlighting its role in biotin biosynthesis and the activity of wild-type BioW with various fatty acid substrates.
Figure 2: Structural biology of BioW.
Figure 3: Native MS analysis of BioW.
Figure 4: Activity of wild-type BioW and active site mutants toward pimelic acid and assays to determine the chain-length specificity of BioW and designed mutants.
Figure 5: Synthesis of heptanoyl-CoA by the engineered BioW Y211F mutant.
Figure 6: Schematic of the BioW active site.

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References

  1. Tong, L. Structure and function of biotin-dependent carboxylases. Cell. Mol. Life Sci. 70, 863–891 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Eisenberg, M.A. The incorporation of 1,7 C14 pimelic acid into biotin vitamers. Biochem. Biophys. Res. Commun. 8, 437–441 (1962).

    Article  CAS  PubMed  Google Scholar 

  3. Eisenberg, M.A. & Krell, K. Synthesis of desthiobiotin from 7,8-diaminopelargonic acid in biotin auxotrophs of Escherichia coli K-12. J. Bacteriol. 98, 1227–1231 (1969).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Eisenberg, M.A. & Star, C. Synthesis of 7-oxo-8-aminopelargonic acid, a biotin vitamer, in cell-free extracts of Escherichia coli biotin auxotrophs. J. Bacteriol. 96, 1291–1297 (1968).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Eisenberg, M.A. & Stoner, G.L. Biosynthesis of 7,8-diaminopelargonic acid, a biotin intermediate, from 7-keto-8-aminopelargonic acid and S-adenosyl-L-methionine. J. Bacteriol. 108, 1135–1140 (1971).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Otsuka, A.J. et al. The Escherichia coli biotin biosynthetic enzyme sequences predicted from the nucleotide sequence of the bio operon. J. Biol. Chem. 263, 19577–19585 (1988).

    Article  CAS  PubMed  Google Scholar 

  7. Lin, S. & Cronan, J.E. Closing in on complete pathways of biotin biosynthesis. Mol. Biosyst. 7, 1811–1821 (2011).

    Article  CAS  PubMed  Google Scholar 

  8. Alexeev, D. et al. The crystal structure of 8-amino-7-oxononanoate synthase: a bacterial PLP-dependent, acyl-CoA-condensing enzyme. J. Mol. Biol. 284, 401–419 (1998).

    Article  CAS  PubMed  Google Scholar 

  9. Alexeev, D., Baxter, R.L. & Sawyer, L. Mechanistic implications and family relationships from the structure of dethiobiotin synthetase. Structure 2, 1061–1072 (1994).

    Article  CAS  PubMed  Google Scholar 

  10. Berkovitch, F., Nicolet, Y., Wan, J.T., Jarrett, J.T. & Drennan, C.L. Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme. Science 303, 76–79 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Huang, W. et al. Crystal structure of an ATP-dependent carboxylase, dethiobiotin synthetase, at 1.65 Å resolution. Structure 2, 407–414 (1994).

    Article  CAS  PubMed  Google Scholar 

  12. Käck, H., Sandmark, J., Gibson, K., Schneider, G. & Lindqvist, Y. Crystal structure of diaminopelargonic acid synthase: evolutionary relationships between pyridoxal-5′-phosphate-dependent enzymes. J. Mol. Biol. 291, 857–876 (1999).

    Article  PubMed  Google Scholar 

  13. Webster, S.P. et al. Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies. Biochemistry 39, 516–528 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Cronan, J.E. & Lin, S. Synthesis of the α,ω-dicarboxylic acid precursor of biotin by the canonical fatty acid biosynthetic pathway. Curr. Opin. Chem. Biol. 15, 407–413 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ifuku, O. et al. Origin of carbon atoms of biotin. 13C-NMR studies on biotin biosynthesis in Escherichia coli. Eur. J. Biochem. 220, 585–591 (1994).

    Article  CAS  PubMed  Google Scholar 

  16. Sanyal, I., Lee, S.-L. & Flint, D.H. Biosynthesis of pimeloyl-CoA, a biotin precursor in Escherichia coli, follows a modified fatty acid synthesis pathway: 13C-labelling studies. J. Am. Chem. Soc. 116, 2637–2638 (1994).

    Article  CAS  Google Scholar 

  17. Lin, S., Hanson, R.E. & Cronan, J.E. Biotin synthesis begins by hijacking the fatty acid synthetic pathway. Nat. Chem. Biol. 6, 682–688 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Agarwal, V., Lin, S., Lukk, T., Nair, S.K. & Cronan, J.E. Structure of the enzyme-acyl carrier protein (ACP) substrate gatekeeper complex required for biotin synthesis. Proc. Natl. Acad. Sci. USA 109, 17406–17411 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bower, S. et al. Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon. J. Bacteriol. 178, 4122–4130 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cryle, M.J. & De Voss, J.J. Carbon–carbon bond cleavage by cytochrome p450(BioI)(CYP107H1). Chem. Commun. (Camb.) 2004, 86–87 (2004).

    Article  Google Scholar 

  21. Cryle, M.J. & Schlichting, I. Structural insights from a P450 carrier protein complex reveal how specificity is achieved in the P450(BioI) ACP complex. Proc. Natl. Acad. Sci. USA 105, 15696–15701 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gloeckler, R. et al. Cloning and characterization of the Bacillus sphaericus genes controlling the bioconversion of pimelate into dethiobiotin. Gene 87, 63–70 (1990).

    Article  CAS  PubMed  Google Scholar 

  23. Ploux, O., Soularue, P., Marquet, A., Gloeckler, R. & Lemoine, Y. Investigation of the first step of biotin biosynthesis in Bacillus sphaericus. Purification and characterization of the pimeloyl-CoA synthase, and uptake of pimelate. Biochem. J. 287, 685–690 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Manandhar, M. & Cronan, J.E. Proofreading of noncognate acyl adenylates by an acyl-coenzyme a ligase. Chem. Biol. 20, 1441–1446 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gulick, A.M. Conformational dynamics in the Acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase. ACS Chem. Biol. 4, 811–827 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Schmelz, S. & Naismith, J.H. Adenylate-forming enzymes. Curr. Opin. Struct. Biol. 19, 666–671 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pendini, N.R. et al. Microbial biotin protein ligases aid in understanding holocarboxylase synthetase deficiency. Biochim. Biophys. Acta 1784, 973–982 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Schmelz, S. et al. AcsD catalyzes enantioselective citrate desymmetrization in siderophore biosynthesis. Nat. Chem. Biol. 5, 174–182 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Finzel, K., Lee, D.J. & Burkart, M.D. Using modern tools to probe the structure–function relationship of fatty acid synthases. ChemBioChem 16, 528–547 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Upson, R.H., Haugland, R.P., Malekzadeh, M.N. & Haugland, R.P. A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate. Anal. Biochem. 243, 41–45 (1996).

    Article  CAS  PubMed  Google Scholar 

  31. Webb, M.R. A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc. Natl. Acad. Sci. USA 89, 4884–4887 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Yount, R.G., Babcock, D., Ballantyne, W. & Ojala, D. Adenylyl imidodiphosphate, an adenosine triphosphate analog containing a P–N–P linkage. Biochemistry 10, 2484–2489 (1971).

    Article  CAS  PubMed  Google Scholar 

  34. Walzthoeni, T., Leitner, A., Stengel, F. & Aebersold, R. Mass spectrometry supported determination of protein complex structure. Curr. Opin. Struct. Biol. 23, 252–260 (2013).

    Article  CAS  PubMed  Google Scholar 

  35. Choi-Rhee, E., Schulman, H. & Cronan, J.E. Promiscuous protein biotinylation by Escherichia coli biotin protein ligase. Protein Sci. 13, 3043–3050 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Tron, C.M. et al. Structural and functional studies of the biotin protein ligase from Aquifex aeolicus reveal a critical role for a conserved residue in target specificity. J. Mol. Biol. 387, 129–146 (2009).

    Article  CAS  PubMed  Google Scholar 

  37. Roux, K.J., Kim, D.I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196, 801–810 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. McElroy, W.D., DeLuca, M. & Travis, J. Molecular uniformity in biological catalyses. The enzymes concerned with firefly luciferin, amino acid, and fatty acid utilization are compared. Science 157, 150–160 (1967).

    Article  CAS  PubMed  Google Scholar 

  39. Huang, Y.T., Lu, S.Y., Yi, C.L. & Lee, C.F. Iron-catalyzed synthesis of thioesters from thiols and aldehydes in water. J. Org. Chem. 79, 4561–4568 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Pal, M. & Bearne, S.L. Synthesis of coenzyme A thioesters using methyl acyl phosphates in an aqueous medium. Org. Biomol. Chem. 12, 9760–9763 (2014).

    Article  CAS  PubMed  Google Scholar 

  41. Beld, J., Finzel, K. & Burkart, M.D. Versatility of acyl-acyl carrier protein synthetases. Chem. Biol. 21, 1293–1299 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Renata, H., Wang, Z.J. & Arnold, F.H. Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution. Angew. Chem. Int. Edn Engl. 54, 3351–3367 (2015).

    Article  CAS  Google Scholar 

  43. Liu, H. & Naismith, J.H. A simple and efficient expression and purification system using two newly constructed vectors. Protein Expr. Purif. 63, 102–111 (2009).

    Article  CAS  PubMed  Google Scholar 

  44. Liu, H. & Naismith, J.H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 8, 91 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Ploux, O. & Marquet, A. The 8-amino-7-oxopelargonate synthase from Bacillus sphaericus. Purification and preliminary characterization of the cloned enzyme overproduced in Escherichia coli. Biochem. J. 283, 327–331 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).

    Article  CAS  Google Scholar 

  47. Zhang, Z., Sauter, N.K., van den Bedem, H., Snell, G. & Deacon, A.M. Automated diffraction image analysis and spot searching for high-throughput crystal screening. J. Appl. Crystallogr. 39, 112–119 (2006).

    Article  CAS  Google Scholar 

  48. Sauter, N.K., Grosse-Kunstleve, R.W. & Adams, P.D. Robust indexing for automatic data collection. J. Appl. Crystallogr. 37, 399–409 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  50. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    Article  CAS  PubMed  Google Scholar 

  51. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  PubMed  Google Scholar 

  52. Skubák, P. & Pannu, N.S. Automatic protein structure solution from weak X-ray data. Nat. Commun. 4, 2777 (2013).

    Article  PubMed  CAS  Google Scholar 

  53. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  CAS  Google Scholar 

  55. Terwilliger, T.C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D Biol. Crystallogr. 64, 61–69 (2008).

    Article  CAS  PubMed  Google Scholar 

  56. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  PubMed  Google Scholar 

  58. Schüttelkopf, A.W. & van Aalten, D.M. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 60, 1355–1363 (2004).

    Article  CAS  PubMed  Google Scholar 

  59. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC, BB/M003493/1) (D.J.C. and J.H.N.) and Wellcome Trust (WT100209MA (J.H.N.)). The native MS data were acquired on an instrument funded by an Engineering and Physical Sciences Research Council (EPSRC) grant to the University of Edinburgh (EP/K039717/1). We acknowledge the use of the Diamond synchrotron. J.H.N. is a Royal Society Wolfson Merit Award Holder and 1000 talent scholar at Sichuan University. M.W. was funded by a University of Edinburgh PhD scholarship. We thank B. Mykhaylyk for help with bioinformatics analysis and L. Mackay for help with MS analysis.

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  1. Menglu Wang and Lucile Moynié: These authors contributed equally to this work.

Authors and Affiliations

  1. EastChem School of Chemistry, University of Edinburgh, Edinburgh, UK

    Menglu Wang, Peter J Harrison, Van Kelly, Andrew Piper & Dominic J Campopiano

  2. Biomedical Sciences Research complex, University of St. Andrews, St. Andrews, UK

    Lucile Moynié & James H Naismith

  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

    James H Naismith

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  1. Menglu Wang
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Contributions

A.P. carried out the initial cloning of the bioW gene and characterization of the recombinant BioW. M.W. and P.J.H. carried out the enzyme isolation, characterization and assay. M.W. generated all the BioW mutants and determined the kinetic parameters of wild-type and mutant enzymes. V.K. performed all protein and acyl-CoA MS analyses. M.W. and L.M. prepared the enzyme for crystal trials and optimized crystallization conditions. L.M. carried out the crystallography experiments with J.H.N. and acquired and interpreted the data. L.M., M.W., P.J.H., V.K., J.H.N. and D.J.C. interpreted the data and wrote the paper.

Corresponding authors

Correspondence to James H Naismith or Dominic J Campopiano.

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Wang, M., Moynié, L., Harrison, P. et al. Using the pimeloyl-CoA synthetase adenylation fold to synthesize fatty acid thioesters. Nat Chem Biol 13, 660–667 (2017). https://doi.org/10.1038/nchembio.2361

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