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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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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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