Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3

Three newly discovered drug metabolizing mutants of cytochrome P450 BM3 (van Vugt‐Lussenburg et al., Identification of critical residues in novel drug metabolizing mutants of Cytochrome P450 BM3 using random mutagenesis, J Med Chem 2007;50:455–461) have been studied at an atomistic level to provide structural explanations for a number of their characteristics. In this study, computational methods are combined with experimental techniques. Molecular dynamics simulations, resonance Raman and UV–VIS spectroscopy, as well as coupling efficiency and substrate‐binding experiments, have been performed. The computational findings, supported by the experimental results, enable structural rationalizations of the mutants. The substrates used in this study are known to be metabolized by human cytochrome P450 2D6. Interestingly, the major metabolites formed by the P450 BM3 mutants differ from those formed by human cytochrome P450 2D6. The computational findings, supported by resonance Raman data, suggest a conformational change of one of the heme propionate groups. The modeling results furthermore suggest that this conformational change allows for an interaction between the negatively charged carboxylate of the heme substituent and the positively charged nitrogen of the substrates. This allows for an orientation of the substrates favorable for formation of the major metabolite by P450 BM3. Proteins 2008. © 2007 Wiley‐Liss, Inc.

[1]  G. V. Paolini,et al.  Empirical scoring functions: I. The development of a fast empirical scoring function to estimate the binding affinity of ligands in receptor complexes , 1997, J. Comput. Aided Mol. Des..

[2]  Chris Oostenbrink,et al.  Metabolic regio- and stereoselectivity of cytochrome P450 2D6 towards 3,4-methylenedioxy-N-alkylamphetamines: in silico predictions and experimental validation. , 2005, Journal of medicinal chemistry.

[3]  Jeffrey P. Jones,et al.  Mechanism of Oxidative Amine Dealkylation of Substituted N,N-Dimethylanilines by Cytochrome P-450: Application of Isotope Effect Profiles , 1995 .

[4]  T. Poulos,et al.  Structure of a cytochrome P450-redox partner electron-transfer complex. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[5]  T. Poulos,et al.  Modeling protein-substrate interactions in the heme domain of cytochrome P450(BM-3). , 1994, Acta crystallographica. Section D, Biological crystallography.

[6]  F. Guengerich,et al.  Cytochrome p450 enzymes in the generation of commercial products , 2002, Nature Reviews Drug Discovery.

[7]  Sudarko,et al.  A survey of active site access channels in cytochromes P450. , 2004, Journal of inorganic biochemistry.

[8]  W U Primrose,et al.  A single mutation in cytochrome P450 BM3 changes substrate orientation in a catalytic intermediate and the regiospecificity of hydroxylation. , 1997, Biochemistry.

[9]  P Willett,et al.  Development and validation of a genetic algorithm for flexible docking. , 1997, Journal of molecular biology.

[10]  Frances H. Arnold,et al.  Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase , 2002, Nature Biotechnology.

[11]  J Deisenhofer,et al.  Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450's. , 1993, Science.

[12]  V. Urlacher,et al.  Microbial P450 enzymes in biotechnology , 2004, Applied Microbiology and Biotechnology.

[13]  H Koga,et al.  Uncoupling of the cytochrome P-450cam monooxygenase reaction by a single mutation, threonine-252 to alanine or valine: possible role of the hydroxy amino acid in oxygen activation. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Victor Guallar,et al.  The role of the heme propionates in heme biochemistry. , 2006, Journal of inorganic biochemistry.

[15]  Yasuhiko Yamamoto,et al.  Control of the redox potential of Pseudomonas aeruginosa cytochrome c551 through the Fe-Met coordination bond strength and pKa of a buried heme propionic acid side chain. , 2005, Biochemistry.

[16]  Dominique Bourgeois,et al.  Structural basis for the mechanism of Ca(2+) activation of the di-heme cytochrome c peroxidase from Pseudomonas nautica 617. , 2004, Structure.

[17]  M. Mewies,et al.  Crystal structure of the ascorbate peroxidase–ascorbate complex , 2003, Nature Structural Biology.

[18]  N. Vermeulen,et al.  Identification of critical residues in novel drug metabolizing mutants of cytochrome P450 BM3 using random mutagenesis. , 2007, Journal of medicinal chemistry.

[19]  L. Narhi,et al.  Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium. , 1986, The Journal of biological chemistry.

[20]  H. Berendsen,et al.  Interaction Models for Water in Relation to Protein Hydration , 1981 .

[21]  Jerome Baudry,et al.  Ile115Leu mutation in the SRS1 region of an insect cytochrome P450 (CYP6B1) compromises substrate turnover via changes in a predicted product release channel. , 2005, Protein engineering, design & selection : PEDS.

[22]  K. R. Marshall,et al.  P450 BM3: the very model of a modern flavocytochrome. , 2002, Trends in biochemical sciences.

[23]  D. van der Spoel,et al.  GROMACS: A message-passing parallel molecular dynamics implementation , 1995 .

[24]  R. Schmid,et al.  Engineering Cytochrome P450 BM-3 for Oxidation of Polycyclic Aromatic Hydrocarbons , 2001, Applied and Environmental Microbiology.

[25]  T. Poulos,et al.  The structure of the cytochrome p450BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid , 1997, Nature Structural Biology.

[26]  R. Wade,et al.  How do substrates enter and products exit the buried active site of cytochrome P450cam? 1. Random expulsion molecular dynamics investigation of ligand access channels and mechanisms. , 2000, Journal of molecular biology.

[27]  J. S. Miles,et al.  Domains of the catalytically self-sufficient cytochrome P-450 BM-3. Genetic construction, overexpression, purification and spectroscopic characterization. , 1992, The Biochemical journal.

[28]  Jonathan P. Clark,et al.  The role of Thr268 and Phe393 in cytochrome P450 BM3. , 2006, Journal of inorganic biochemistry.

[29]  J. S. Miles,et al.  The role of tryptophan 97 of cytochrome P450 BM3 from Bacillus megaterium in catalytic function. Evidence against the 'covalent switching' hypothesis of P-450 electron transfer. , 1994, The Biochemical journal.

[30]  A. Munro,et al.  Roles of key active-site residues in flavocytochrome P450 BM3. , 1999, The Biochemical journal.

[31]  F. Arnold,et al.  Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450. , 2006, Biotechnology and bioengineering.

[32]  N. Vermeulen,et al.  Heterotropic and homotropic cooperativity by a drug-metabolising mutant of cytochrome P450 BM3. , 2006, Biochemical and biophysical research communications.

[33]  C. Slaughter,et al.  Fatty acid monooxygenation by P450BM-3: product identification and proposed mechanisms for the sequential hydroxylation reactions. , 1992, Archives of biochemistry and biophysics.

[34]  S. Martinis,et al.  Crystal structure of the cytochrome P-450CAM active site mutant Thr252Ala. , 1991, Biochemistry.

[35]  R. Schmid,et al.  Residue size at position 87 of cytochrome P450 BM‐3 determines its stereoselectivity in propylbenzene and 3‐chlorostyrene oxidation , 2001, FEBS letters.

[36]  R D Schmid,et al.  Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst. , 2000, Chemistry.

[37]  T. Ost,et al.  Phe393 mutants of cytochrome P450 BM3 with modified heme redox potentials have altered heme vinyl and propionate conformations. , 2004, Biochemistry.

[38]  L. Wong,et al.  Protein engineering of Bacillus megaterium CYP102. The oxidation of polycyclic aromatic hydrocarbons. , 2001, European journal of biochemistry.

[39]  R D Schmid,et al.  Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant. , 2001, Biochimica et biophysica acta.

[40]  S. Boddupalli,et al.  Fatty acid monooxygenation by cytochrome P-450BM-3. , 1990, The Journal of biological chemistry.

[41]  L. Waskell,et al.  The Stoichiometry of the Cytochrome P-450-catalyzed Metabolism of Methoxyflurane and Benzphetamine in the Presence and Absence of Cytochrome b5(*) , 1995, The Journal of Biological Chemistry.

[42]  G. Ciccotti,et al.  Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes , 1977 .

[43]  Wilfred F. van Gunsteren,et al.  A generalized reaction field method for molecular dynamics simulations , 1995 .

[44]  M. Machius,et al.  Pivotal role of water in the mechanism of P450BM-3. , 2001, Biochemistry.

[45]  Markus Christen,et al.  The GROMOS software for biomolecular simulation: GROMOS05 , 2005, J. Comput. Chem..

[46]  H. Berendsen,et al.  Molecular dynamics with coupling to an external bath , 1984 .

[47]  T. Poulos,et al.  The role of Thr268 in oxygen activation of cytochrome P450BM-3. , 1995, Biochemistry.

[48]  Wilfred F. van Gunsteren,et al.  An improved GROMOS96 force field for aliphatic hydrocarbons in the condensed phase , 2001, J. Comput. Chem..

[49]  M. Sutcliffe,et al.  The catalytic mechanism of cytochrome P450 BM3 involves a 6 Å movement of the bound substrate on reduction , 1996, Nature Structural Biology.

[50]  B. Robert.  Resonance Raman spectroscopy , 2009, Photosynthesis Research.

[51]  T. Ost,et al.  Phenylalanine 393 exerts thermodynamic control over the heme of flavocytochrome P450 BM3. , 2001, Biochemistry.

[52]  Rebecca C Wade,et al.  The ins and outs of cytochrome P450s. , 2007, Biochimica et biophysica acta.

[53]  Alan E. Mark,et al.  The GROMOS96 Manual and User Guide , 1996 .

[54]  Xiaoyuan Li,et al.  Resonance Raman Spectroscopy of Metalloporphyrins , 1988 .

[55]  P. Hollenberg,et al.  Comparison of substrate metabolism by cytochromes P450 2B1, 2B4, and 2B6: relationship of heme spin state, catalysis, and the effects of cytochrome b5. , 2003, Journal of inorganic biochemistry.

[56]  M. Berenbaum,et al.  Identification of variable amino acids in the SRS1 region of CYP6B1 modulating furanocoumarin metabolism. , 2004, Archives of biochemistry and biophysics.

[57]  Christopher W. Murray,et al.  Empirical scoring functions. II. The testing of an empirical scoring function for the prediction of ligand-receptor binding affinities and the use of Bayesian regression to improve the quality of the model , 1998, J. Comput. Aided Mol. Des..

[58]  Chris Oostenbrink,et al.  Catalytic site prediction and virtual screening of cytochrome P450 2D6 substrates by consideration of water and rescoring in automated docking. , 2006, Journal of medicinal chemistry.

[59]  Berk Hess,et al.  GROMACS 3.0: a package for molecular simulation and trajectory analysis , 2001 .

[60]  V. Urlacher,et al.  Immobilisation of P450 BM‐3 and an NADP+ Cofactor Recycling System: Towards a Technical Application of Heme‐Containing Monooxygenases in Fine Chemical Synthesis , 2003 .

[61]  M. Joyce,et al.  A Single Mutation in Cytochrome P450 BM3 Induces the Conformational Rearrangement Seen upon Substrate Binding in the Wild-type Enzyme* , 2004, Journal of Biological Chemistry.