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The role of distant mutations and allosteric regulation on LovD active site dynamics

Nature Chemical Biology volume 10, pages 431–436 (2014)Cite this article

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Abstract

Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1,000-fold more efficient in the synthesis of the drug simvastatin than the wild-type LovD. This is to our knowledge the first nonpatent report of the enzyme currently used for the manufacture of simvastatin as well as the intermediate evolved variants. Crystal structures and microsecond-scale molecular dynamics simulations revealed the mechanism by which the laboratory-generated mutations free LovD from dependence on protein-protein interactions. Mutations markedly altered conformational dynamics of the catalytic residues, obviating the need for allosteric modulation by the acyl carrier LovF.

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Figure 1: Chemical reactions catalyzed by LovD.
Figure 2: Location and structural effects of laboratory-evolved mutations in LovD.
Figure 3: Structural features of the Ser76-Lys79-Tyr188 catalytic triad.
Figure 4: Active site dynamics of LovD variants determined through MD simulations.
Figure 5: Conformational ensembles of the active site of LovD variants obtained through MD simulations.
Figure 6: The role of protein-protein interactions on LovD active site dynamics.

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NCBI Reference Sequence

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References

  1. Arnold, F.H. & Volkov, A.A. Directed evolution of biocatalysts. Curr. Opin. Chem. Biol. 3, 54–59 (1999).

    Article  CAS  Google Scholar 

  2. Benkovic, S.J. & Hammes-Schiffer, S. A perspective on enzyme catalysis. Science 301, 1196–1202 (2003).

    Article  CAS  Google Scholar 

  3. Kiss, G., Çelebi-Ölçüm, N., Moretti, R., Baker, D. & Houk, K.N. Computational enzyme design. Angew. Chem. Int. Ed. Engl. 52, 5700–5725 (2013).

    Article  CAS  Google Scholar 

  4. Kennedy, J. et al. Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science 284, 1368–1372 (1999).

    Article  CAS  Google Scholar 

  5. Xie, X., Meehan, M.J., Xu, W., Dorrestein, P.C. & Tang, Y. Acyltransferase mediated polyketide release from a fungal megasynthase. J. Am. Chem. Soc. 131, 8388–8389 (2009).

    Article  CAS  Google Scholar 

  6. Xie, X. & Tang, Y. Efficient synthesis of simvastatin by use of whole-cell biocatalysis. Appl. Environ. Microbiol. 73, 2054–2060 (2007).

    Article  CAS  Google Scholar 

  7. Gao, X. et al. Directed evolution and structural characterization of a simvastatin synthase. Chem. Biol. 16, 1064–1074 (2009).

    Article  CAS  Google Scholar 

  8. Fox, R.J. et al. Improving catalytic function by ProSAR-driven enzyme evolution. Nat. Biotechnol. 25, 338–344 (2007).

    Article  CAS  Google Scholar 

  9. Gilson, L. et al. Variant LovD polypeptides and their uses. World Intellectual Property Organization patent WO2011/041231 (2011).

  10. Collier, S.J., Teo, E.L., Sukurmaran, J., Wilson, R.J. & Xu, J. Improved LovD acyltransferase mediated acylation. World Intellectual Patent Organization patent WO2011/041233 (2011).

  11. Savile, C.K. et al. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329, 305–309 (2010).

    Article  CAS  Google Scholar 

  12. Shan, Y., Arkhipov, A., Kim, E.T., Pan, A.C. & Shaw, D.E. Transitions to catalytically inactive conformations in EGFR kinase. Proc. Natl. Acad. Sci. USA 110, 7270–7275 (2013).

    Article  CAS  Google Scholar 

  13. Bhabha, G. et al. A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis. Science 332, 234–238 (2011).

    Article  CAS  Google Scholar 

  14. Shaw, D.E. et al. Anton, a special-purpose machine for molecular dynamics simulation. Commun. ACM 51, 91–97 (2008).

    Article  Google Scholar 

  15. Lindorff-Larsen, K., Piana, S., Dror, R.O. & Shaw, D.E. How fast-folding proteins fold. Science 334, 517–520 (2011).

    Article  CAS  Google Scholar 

  16. Shaw, D.E. et al. Atomic-level characterization of the structural dynamics of proteins. Science 330, 341–346 (2010).

    Article  CAS  Google Scholar 

  17. Maier, T., Jenni, S. & Ban, N. Architecture of mammalian fatty acid synthase at 4.5 Å resolution. Science 311, 1258–1262 (2006).

    Article  CAS  Google Scholar 

  18. Jenni, S. et al. Structure of fungal fatty acid synthase and implications for iterative substrate shuttling. Science 316, 254–261 (2007).

    Article  CAS  Google Scholar 

  19. 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  Google Scholar 

  20. Wagner, U.G., Petersen, E.I., Schwab, H. & Kratky, C. EstB from Burkholderia gladioli: a novel esterase with a β-lactamase fold reveals steric factors to discriminate between esterolytic and β-lactam cleaving activity. Protein Sci. 11, 467–478 (2002).

    Article  CAS  Google Scholar 

  21. Adamczyk, A.J., Cao, J., Kamerlin, S.C.L. & Warshel, A. Catalysis by dihydrofolate reductase and other enzymes arises from electrostatic preorganization, not conformational motions. Proc. Natl. Acad. Sci. USA 108, 14115–14120 (2011).

    Article  CAS  Google Scholar 

  22. Schulenburg, C. & Hilvert, D. in Dynamics in Enzyme Catalysis (eds. Klinman, J. & Hammes-Schiffer, S.) 41–67 (Springer Berlin Heidelberg, 2013).

  23. Wong, K.F., Selzer, T., Benkovic, S.J. & Hammes-Schiffer, S. Impact of distal mutations on the network of coupled motions correlated to hydride transfer in dihydrofolate reductase. Proc. Natl. Acad. Sci. USA 102, 6807–6812 (2005).

    Article  CAS  Google Scholar 

  24. Coelho, P.S., Brustad, E.M., Kannan, A. & Arnold, F.H. Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339, 307–310 (2013).

    Article  CAS  Google Scholar 

  25. Davis, S., Huisman, G., Jenne, S., Krebber, A. & Newman, L. Ketoreductase polypeptides and related polynucleotides. US Patent US2006/0195947 (2006).

  26. Colbeck, J. et al. Combined automated parallel synthesis of polynucleotide variants. US Patent US8383346 (2013).

  27. Fox, R.J. et al. Improving catalytic function by ProSAR-driven enzyme evolution. Nat. Biotechnol. 25, 338–344 (2007).

    Article  CAS  Google Scholar 

  28. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. 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  Google Scholar 

  31. Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D Biol. Crystallogr. 60, 2210–2221 (2004).

    Article  CAS  Google Scholar 

  32. Winn, M.D., Murshudov, G.N. & Papiz, M.Z. Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300–321 (2003).

    Article  CAS  Google Scholar 

  33. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of COOT. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  34. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  35. Colovos, C. & Yeates, T.O. Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci. 2, 1511–1519 (1993).

    Article  CAS  Google Scholar 

  36. Lüthy, R., Bowie, J.U. & Eisenberg, D. Assessment of protein models with three-dimensional profiles. Nature 356, 83–85 (1992).

    Article  Google Scholar 

  37. Zhao, Y. & Truhlar, D. The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals. Theor. Chem. Acc. 120, 215–241 (2008).

    Article  CAS  Google Scholar 

  38. Scalmani, G. & Frisch, M.J. Continuous surface charge polarizable continuum models of solvation. I. General formalism. J. Chem. Phys. 132, 114110 (2010).

    Article  Google Scholar 

  39. Schutz, C.N. & Warshel, A. What are the dielectric “constants” of proteins and how to validate electrostatic models? Proteins 44, 400–417 (2001).

    Article  CAS  Google Scholar 

  40. Li, L., Li, C., Zhang, Z. & Alexov, E. On the dielectric “constant” of proteins: smooth dielectric function for macromolecular modeling and its implementation in Delphi. J. Chem. Theory Comput. 9, 2126–2136 (2013).

    Article  CAS  Google Scholar 

  41. Wattana-Amorn, P. et al. Solution structure of an acyl carrier protein domain from a fungal type I polyketide synthase. Biochemistry 49, 2186–2193 (2010).

    Article  CAS  Google Scholar 

  42. Kozakov, D. et al. Achieving reliability and high accuracy in automated protein docking: Cluspro, PIPER, SDU, and stability analysis in CAPRI rounds 13–19. Proteins. 78, 3124–3130 (2010).

    Article  CAS  Google Scholar 

  43. Kozakov, D., Brenke, R., Comeau, S.R. & Vajda, S. PIPER: an FFT-based protein docking program with pairwise potentials. Proteins 65, 392–406 (2006).

    Article  CAS  Google Scholar 

  44. Comeau, S.R., Gatchell, D.W., Vajda, S. & Camacho, C.J. ClusPro: an automated docking and discrimination method for the prediction of protein complexes. Bioinformatics 20, 45–50 (2004).

    Article  CAS  Google Scholar 

  45. Comeau, S.R., Gatchell, D.W., Vajda, S. & Camacho, C.J. ClusPro: a fully automated algorithm for protein–protein docking. Nucleic Acids Res. 32, W96–W99 (2004).

    Article  CAS  Google Scholar 

  46. Lyskov, S. & Gray, J.J. The RosettaDock server for local protein-protein docking. Nucleic Acids Res. 36, W233–W238 (2008).

    Article  CAS  Google Scholar 

  47. Verdonk, M.L., Cole, J.C., Hartshorn, M.J., Murray, C.W. & Taylor, R.D. Improved protein-ligand docking using GOLD. Proteins 52, 609–623 (2003).

    Article  CAS  Google Scholar 

  48. Richter, F., Leaver-Fay, A., Khare, S.D., Bjelic, S. & Baker, D. De novo enzyme design using Rosetta3. PLoS ONE 6, e19230 (2011).

    Article  CAS  Google Scholar 

  49. Wang, J., Wolf, R.M., Caldwell, J.W., Kollman, P.A. & Case, D.A. Development and testing of a general Amber force field. J. Comput. Chem. 25, 1157–1174 (2004).

    Article  CAS  Google Scholar 

  50. Bayly, C.I., Cieplak, P., Cornell, W. & Kollman, P.A. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem. 97, 10269–10280 (1993).

    Article  CAS  Google Scholar 

  51. Besler, B.H., Merz, K.M. & Kollman, P.A. Atomic charges derived from semiempirical methods. J. Comput. Chem. 11, 431–439 (1990).

    Article  CAS  Google Scholar 

  52. Singh, U.C. & Kollman, P.A. An approach to computing electrostatic charges for molecules. J. Comput. Chem. 5, 129–145 (1984).

    Article  CAS  Google Scholar 

  53. Jorgensen, W.L., Chandrasekhar, J., Madura, J.D., Impey, R.W. & Klein, M.L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).

    Article  CAS  Google Scholar 

  54. Wang, J., Cieplak, P. & Kollman, P.A. How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J. Comput. Chem. 21, 1049–1074 (2000).

    Article  CAS  Google Scholar 

  55. Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: an N-log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10093 (1993).

    Article  CAS  Google Scholar 

  56. Shan, Y., Klepeis, J.L., Eastwood, M.P., Dror, R.O. & Shaw, D.E. Gaussian split Ewald: a fast Ewald mesh method for molecular simulation. J. Chem. Phys. 122, 54101 (2005).

    Article  Google Scholar 

  57. Essmann, U. et al. A smooth particle mesh Ewald method. J. Chem. Phys. 103, 8577 (1995).

    Article  CAS  Google Scholar 

  58. Tuckerman, M., Berne, B.J. & Martyna, G.J. Reversible multiple time scale molecular dynamics. J. Chem. Phys. 97, 1990 (1992).

    CAS  Google Scholar 

  59. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. Model. 14, 33–38 (1996).

    Article  CAS  Google Scholar 

  60. Voss, N.R., Gerstein, M., Steitz, T.A. & Moore, P.B. The geometry of the ribosomal polypeptide exit tunnel. J. Mol. Biol. 360, 893–906 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was funded by the National Institutes of Health through grant 1R01GM097200 (to Y.T., T.O.Y. and K.N.H.) and GM075962 (to K.N.H.). G.J.-O. and S.O. were supported by Ministerio de Economía y Competitividad (EX2012-1063) and European Union Marie Curie (PIOF-GA-2009-252856) postdoctoral fellowships, respectively. We thank the LovD evolution team at Codexis, Inc. for generating the variants used for this work. ANTON simulations were performed on the National Resource for Biomedical Supercomputing at the Pittsburgh Supercomputing Centre with funding from the National Institute of General Medical Sciences under grant RC2GM093307. Additional calculations were performed on the MinoTauro cluster at the Barcelona Supercomputing Center and Hoffman2 and Dawson2 graphics processing unit clusters at the University of California–Los Angeles and the Extreme Science and Engineering Discovery Environment, which is supported by the National Science Foundation (OCI-1053575).

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Author notes

  1. Gonzalo Jiménez-Osés, Sílvia Osuna and Xue Gao: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California–Los Angeles, Los Angeles, California, USA

    Gonzalo Jiménez-Osés, Sílvia Osuna, Yi Tang & K N Houk

  2. Department of Chemical and Biomolecular Engineering, University of California–Los Angeles, Los Angeles, California, USA

    Xue Gao & Yi Tang

  3. Molecular Biology Institute, University of California–Los Angeles, Los Angeles, California, USA

    Michael R Sawaya & Todd O Yeates

  4. Codexis, Inc., Redwood City, California, USA

    Lynne Gilson, Steven J Collier & Gjalt W Huisman

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  1. Gonzalo Jiménez-Osés
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Contributions

K.N.H., Y.T., G.W.H. and T.O.Y. planned research; L.G. and S.J.C. planned directed evolution experiments; X.G. and M.R.S. did crystallography; X.G. did kinetics, G.J.-O. and S.O. performed MD simulations; all of the authors analyzed data; G.J.-O., S.O., K.N.H., Y.T., T.O.Y. and G.W.H. wrote paper.

Corresponding authors

Correspondence to Gjalt W Huisman, Todd O Yeates, Yi Tang or K N Houk.

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Competing interests

L.G. and S.J.C. are shareholders in Codexis. G.W.H. is a Codexis employee and holds shares and stock options in Codexis.

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Supplementary Results, Supplementary Figures 1–17 and Supplementary Table 1. (PDF 10434 kb)

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Jiménez-Osés, G., Osuna, S., Gao, X. et al. The role of distant mutations and allosteric regulation on LovD active site dynamics. Nat Chem Biol 10, 431–436 (2014). https://doi.org/10.1038/nchembio.1503

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