Expanding the toolbox of organic chemists: directed evolution of P450 monooxygenases as catalysts in regio- and stereoselective oxidative hydroxylation.

Cytochrome P450 enzymes (CYPs) have been used for more than six decades as catalysts for the CH-activating oxidative hydroxylation of organic compounds with formation of added-value products. However, it has not been possible to control regio- and stereoselectivity in a general manner, which is necessary for wide (industrial) applications. Especially directed evolution as a Darwinian approach to protein engineering has changed the situation for the better during the last 3-4 years leading to extensive progress, which is summarized and analysed in this Feature Article.

[1]  V. Urlacher,et al.  Cluster Screening: An Effective Approach for Probing the Substrate Space of Uncharacterized Cytochrome P450s , 2013, Chembiochem : a European journal of chemical biology.

[2]  Manfred T. Reetz,et al.  Directed Evolution of an Enantioselective Enzyme through Combinatorial Multiple-Cassette Mutagenesis. , 2001, Angewandte Chemie.

[3]  D. Sherman,et al.  Selective oxidation of carbolide C–H bonds by an engineered macrolide P450 mono-oxygenase , 2009, Proceedings of the National Academy of Sciences.

[4]  Frank Schulz,et al.  Minimally invasive mutagenesis gives rise to a biosynthetic polyketide library. , 2012, Angewandte Chemie.

[5]  Frances H Arnold,et al.  Chemo-enzymatic fluorination of unactivated organic compounds. , 2009, Nature chemical biology.

[6]  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.

[7]  Adrian J Mulholland,et al.  Compound I reactivity defines alkene oxidation selectivity in cytochrome P450cam. , 2010, The journal of physical chemistry. B.

[8]  Y Fujii-Kuriyama,et al.  Site-directed mutageneses of rat liver cytochrome P-450d: catalytic activities toward benzphetamine and 7-ethoxycoumarin. , 1989, Biochemistry.

[9]  Joelle N. Pelletier,et al.  Expanding the organic toolbox: a guide to integrating biocatalysis in synthesis. , 2012, Chemical Society reviews.

[10]  Jizhen Zhang,et al.  Catalytic oxidation of light alkanes (C1-C4) by heteropoly compounds. , 2014, Chemical reviews.

[11]  B. Trost,et al.  Asymmetric transition-metal-catalyzed allylic alkylations: applications in total synthesis. , 2003, Chemical reviews.

[12]  J. Pleiss,et al.  Conservation analysis of class‐specific positions in cytochrome P450 monooxygenases: Functional and structural relevance , 2014, Proteins.

[13]  S. Bell,et al.  Selective aliphatic carbon-hydrogen bond activation of protected alcohol substrates by cytochrome P450 enzymes. , 2014, Organic & biomolecular chemistry.

[14]  Vlada B Urlacher,et al.  Screening of a minimal enriched P450 BM3 mutant library for hydroxylation of cyclic and acyclic alkanes. , 2011, Chemical communications.

[15]  K. Auclair,et al.  Controlling substrate specificity and product regio- and stereo-selectivities of P450 enzymes without mutagenesis. , 2014, Bioorganic & medicinal chemistry.

[16]  Joseph P. Adams,et al.  Asymmetric epoxidation of alkenes and benzylic hydroxylation with P450tol monooxygenase from Rhodococcus coprophilus TC-2. , 2014, Chemical communications.

[17]  P. R. Montellano Hydrocarbon hydroxylation by cytochrome P450 enzymes. , 2010 .

[18]  U. Schwaneberg,et al.  First steps towards a Zn/Co(III)sep-driven P450 BM3 reactor , 2011, Applied Microbiology and Biotechnology.

[19]  Manfred T Reetz,et al.  Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution , 2011, Nature Chemistry.

[20]  V. Urlacher,et al.  A novel P450-based biocatalyst for the selective production of chiral 2-alkanols. , 2014, Chemical Communications.

[21]  S. Bell,et al.  A Highly Active Single‐Mutation Variant of P450BM3 (CYP102A1) , 2009, Chembiochem : a European journal of chemical biology.

[22]  S. Bell,et al.  P450(BM3) (CYP102A1): connecting the dots. , 2012, Chemical Society reviews.

[23]  Jullien Drone,et al.  A regioselective biocatalyst for alkane activation under mild conditions. , 2011, Angewandte Chemie.

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

[25]  V. Sieber,et al.  Biocatalytic Synthesis of a Diketobornane as a Building Block for Bifunctional Camphor Derivatives , 2013 .

[26]  M. Reetz,et al.  Directed Evolution of an Enantioselective Enoate-Reductase: Testing the Utility of Iterative Saturation Mutagenesis , 2009 .

[27]  Philip A. Romero,et al.  Exploring protein fitness landscapes by directed evolution , 2009, Nature Reviews Molecular Cell Biology.

[28]  I. Arends,et al.  The taming of oxygen: biocatalytic oxyfunctionalisations. , 2014, Chemical communications.

[29]  S. D. Beer,et al.  Regio‐ and Stereoselective Hydroxylation of Optically Active α‐Ionone Enantiomers by Engineered Cytochrome P450 BM3 Mutants , 2012 .

[30]  S. Laschat,et al.  Selective allylic hydroxylation of acyclic terpenoids by CYP154E1 from Thermobifida fusca YX , 2014, Beilstein journal of organic chemistry.

[31]  Kurt Faber,et al.  Biotransformations in Organic Chemistry , 1992 .

[32]  M. Reetz,et al.  Biocatalysis in organic chemistry and biotechnology: past, present, and future. , 2013, Journal of the American Chemical Society.

[33]  V. Urlacher,et al.  Novel family members of CYP109 from Sorangium cellulosum So ce56 exhibit characteristic biochemical and biophysical properties , 2013, Biotechnology and applied biochemistry.

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

[35]  Manfred T Reetz,et al.  Tuning a p450 enzyme for methane oxidation. , 2011, Angewandte Chemie.

[36]  R. Agudo,et al.  Stereo- and regioselectivity in the P450-catalyzed oxidative tandem difunctionalization of 1-methylcyclohexene , 2013 .

[37]  R. Lonsdale,et al.  CH-activating oxidative hydroxylation of 1-tetralones and related compounds with high regio- and stereoselectivity. , 2014, Chemical communications.

[38]  Weng Lin Tang,et al.  High-throughput method for determining the enantioselectivity of enzyme-catalyzed hydroxylations based on mass spectrometry. , 2010, Angewandte Chemie.

[39]  S. S. Yu,et al.  Regioselective hydroxylation of C(12)-C(15) fatty acids with fluorinated substituents by cytochrome P450 BM3. , 2013, Chemistry.

[40]  Jeremy N. Harvey,et al.  Inclusion of Dispersion Effects Significantly Improves Accuracy of Calculated Reaction Barriers for Cytochrome P450 Catalyzed Reactions , 2010 .

[41]  G. Gilardi,et al.  Identification of mutant Asp251Gly/Gln307His of cytochrome P450 BM3 for the generation of metabolites of diclofenac, ibuprofen and tolbutamide. , 2012, Chemistry.

[42]  Bernhard Hauer,et al.  An Efficient Route to Selective Bio‐oxidation Catalysts: an Iterative Approach Comprising Modeling, Diversification, and Screening, Based on CYP102A1 , 2011, Chembiochem : a European journal of chemical biology.

[43]  J. Pietruszka,et al.  Enantioselective allylic hydroxylation of ω-alkenoic acids and esters by P450 BM3 monooxygenase. , 2014, Angewandte Chemie.

[44]  D. Zheng,et al.  Regio- and stereoselective benzylic hydroxylation to synthesize chiral tetrahydroquinolin-4-ol and tetrahydro-1H-benzo[b]azepin-5-ol with Pseudomonas plecoglossicidas , 2014 .

[45]  M. Reetz,et al.  Directed evolution of cyclohexanone monooxygenases: enantioselective biocatalysts for the oxidation of prochiral thioethers. , 2004, Angewandte Chemie.

[46]  Manfred T Reetz,et al.  Laboratory evolution of stereoselective enzymes: a prolific source of catalysts for asymmetric reactions. , 2011, Angewandte Chemie.

[47]  F. Arnold,et al.  Combinatorial Alanine Substitution Enables Rapid Optimization of Cytochrome P450BM3 for Selective Hydroxylation of Large Substrates , 2010, Chembiochem : a European journal of chemical biology.

[48]  H. Pellissier,et al.  CHEMICAL AND BIOCHEMICAL HYDROXYLATIONS OF STEROIDS. A REVIEW , 2001 .

[49]  Manfred T. Reetz,et al.  Creation of Enantioselective Biocatalysts for Organic Chemistry by In Vitro Evolution , 1997 .

[50]  Vlada B Urlacher,et al.  Light‐driven biocatalysis with cytochrome P450 peroxygenases , 2013, Biotechnology and applied biochemistry.

[51]  Christian Wandrey,et al.  Industrial Biotransformations: LIESE: INDUSTRIAL BIOTRANSFORMATIONS O-BK , 2006 .

[52]  Linda G. Otten,et al.  Enzyme engineering for enantioselectivity: from trial-and-error to rational design? , 2010, Trends in biotechnology.

[53]  Zhanglin Lin,et al.  Semi‐rational engineering of cytochrome P450sca‐2 in a hybrid system for enhanced catalytic activity: Insights into the important role of electron transfer , 2013, Biotechnology and bioengineering.

[54]  Jürgen Pleiss,et al.  Multiple molecular dynamics simulations of human p450 monooxygenase CYP2C9: The molecular basis of substrate binding and regioselectivity toward warfarin , 2006, Proteins.

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

[56]  Wei Li,et al.  New α-Tetralonyl Glucosides from the Fruit of Juglans mandshurica , 2004 .

[57]  Nicholas J. Turner,et al.  Substrate promiscuity of cytochrome P450 RhF , 2013 .

[58]  H. Bohets,et al.  Recombinant Escherichia coli cells immobilized in Ca-alginate beads for metabolite production , 2009 .

[59]  N. Vermeulen,et al.  Evaluation of alkoxyresorufins as fluorescent substrates for high-throughput screening of cytochrome P450BM3 and site-directed mutants , 2005 .

[60]  M. White Adding Aliphatic C–H Bond Oxidations to Synthesis , 2012, Science.

[61]  P. Baran,et al.  If C-H bonds could talk: selective C-H bond oxidation. , 2011, Angewandte Chemie.

[62]  Yong Wang,et al.  P450 enzymes: their structure, reactivity, and selectivity-modeled by QM/MM calculations. , 2010, Chemical reviews.

[63]  G. Sprenger,et al.  Selective Catalytic Oxidation of CH Bonds with Molecular Oxygen , 2013 .

[64]  Harry A. Stern,et al.  Controlled Oxidation of Remote sp3 C–H Bonds in Artemisinin via P450 Catalysts with Fine-Tuned Regio- and Stereoselectivity , 2012, Journal of the American Chemical Society.

[65]  J. Woodley,et al.  Guidelines and Cost Analysis for Catalyst Production in Biocatalytic Processes , 2011 .

[66]  Huilei Yu,et al.  Unusually Broad Substrate Profile of Self‐Sufficient Cytochrome P450 Monooxygenase CYP116B4 from Labrenzia aggregata , 2014, Chembiochem : a European journal of chemical biology.

[67]  X. Ribas,et al.  The Iron(II) Complex [Fe(CF3SO3)2(mcp)] as a Convenient, Readily Available Catalyst for the Selective Oxidation of Methylenic Sites in Alkanes , 2014 .

[68]  Zhi Li,et al.  Engineering of p450pyr hydroxylase for the highly regio- and enantioselective subterminal hydroxylation of alkanes. , 2014, Angewandte Chemie.

[69]  Yosephine Gumulya,et al.  Many Pathways in Laboratory Evolution Can Lead to Improved Enzymes: How to Escape from Local Minima , 2012, Chembiochem : a European journal of chemical biology.

[70]  F. Arnold,et al.  Enantioselective alpha-hydroxylation of 2-arylacetic acid derivatives and buspirone catalyzed by engineered cytochrome P450 BM-3. , 2006, Journal of the American Chemical Society.

[71]  David Taub,et al.  The Total Synthesis of Steroids1 , 1952 .

[72]  Manfred T. Reetz,et al.  A GC-based method for high-throughput screening of enantioselective catalysts , 2001 .

[73]  M. Reetz,et al.  Light‐Driven Biocatalytic Oxidation and Reduction Reactions: Scope and Limitations , 2008, Chembiochem : a European journal of chemical biology.

[74]  Karl H. Clodfelter,et al.  Computational solvent mapping reveals the importance of local conformational changes for broad substrate specificity in mammalian cytochromes P450. , 2006, Biochemistry.

[75]  Rudi Fasan,et al.  Enhancing the Efficiency and Regioselectivity of P450 Oxidation Catalysts by Unnatural Amino Acid Mutagenesis , 2014, Chembiochem : a European journal of chemical biology.

[76]  Bernhard Hauer,et al.  New generation of biocatalysts for organic synthesis. , 2014, Angewandte Chemie.

[77]  Gheorghe-Doru Roiban,et al.  Cytochrome P450 catalyzed oxidative hydroxylation of achiral organic compounds with simultaneous creation of two chirality centers in a single C-H activation step. , 2014, Angewandte Chemie.

[78]  T. Darden,et al.  Altering the regiospecificity of androstenedione hydroxylase activity in P450s 2a-4/5 by a mutation of the residue at position 481. , 1995, Biochemistry.

[79]  G. A. Berchtold,et al.  Total synthesis of (-)-chorismic acid and (-)-shikimic acid , 1987 .

[80]  M. Reetz,et al.  Designer cells for stereocomplementary de novo enzymatic cascade reactions based on laboratory evolution. , 2013, Chemical communications.

[81]  Jan Marienhagen,et al.  Direct oxidation of cycloalkanes to cycloalkanones with oxygen in water. , 2013, Angewandte Chemie.

[82]  Frank Hollmann,et al.  Biocatalytic Redox Reactions for Organic Synthesis: Nonconventional Regeneration Methods , 2010 .

[83]  J. Hogg Steroids, the steroid community, and Upjohn in perspective: a profile of innovation , 1992, Steroids.

[84]  S. Wijmenga,et al.  Active site substitution A82W improves the regioselectivity of steroid hydroxylation by cytochrome P450 BM3 mutants as rationalized by spin relaxation nuclear magnetic resonance studies. , 2012, Biochemistry.

[85]  Wayne M Patrick,et al.  Strategies and computational tools for improving randomized protein libraries. , 2005, Biomolecular engineering.

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

[87]  Oliver May,et al.  Application of designed enzymes in organic synthesis. , 2011, Chemical reviews.

[88]  Nicholas J Turner,et al.  Directed evolution of an amine oxidase possessing both broad substrate specificity and high enantioselectivity. , 2003, Angewandte Chemie.

[89]  Matthias Dietrich,et al.  Altering the regioselectivity of the subterminal fatty acid hydroxylase P450 BM-3 towards gamma- and delta-positions. , 2009, Journal of biotechnology.

[90]  Nicholas J Turner,et al.  Directed evolution drives the next generation of biocatalysts. , 2009, Nature chemical biology.

[91]  Karlheinz Drauz,et al.  Enzyme Catalysis in Organic Synthesis , 1995 .

[92]  Rudi Fasan,et al.  P450 fingerprinting method for rapid discovery of terpene hydroxylating P450 catalysts with diversified regioselectivity. , 2011, Journal of the American Chemical Society.

[93]  A. Ilie,et al.  P450-catalyzed regio- and stereoselective oxidative hydroxylation of disubstituted cyclohexanes: creation of three centers of chirality in a single CH-activation event , 2015 .

[94]  S. Sligar,et al.  Decreased substrate affinity upon alteration of the substrate‐docking region in cytochrome P450BM‐3 , 1997, FEBS letters.

[95]  F Peter Guengerich,et al.  Complex reactions catalyzed by cytochrome P450 enzymes. , 2007, Biochimica et biophysica acta.

[96]  F. Arnold,et al.  Enzymatic functionalization of carbon-hydrogen bonds. , 2011, Chemical Society reviews.

[97]  R. Bernhardt,et al.  Changing the Regioselectivity of a P450 from C15 to C11 Hydroxylation of Progesterone , 2012, Chembiochem : a European journal of chemical biology.

[98]  Frances H Arnold,et al.  Engineered alkane-hydroxylating cytochrome P450(BM3) exhibiting nativelike catalytic properties. , 2007, Angewandte Chemie.

[99]  M. Machius,et al.  Crystal structure of inhibitor-bound P450BM-3 reveals open conformation of substrate access channel. , 2008, Biochemistry.

[100]  Manfred T Reetz,et al.  The importance of additive and non-additive mutational effects in protein engineering. , 2013, Angewandte Chemie.

[101]  D. Sherman,et al.  Diversity of P450 enzymes in the biosynthesis of natural products. , 2012, Natural product reports.

[102]  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.

[103]  R. Fasan Tuning P450 Enzymes as Oxidation Catalysts , 2012 .

[104]  Lionel Cheruzel,et al.  Regio- and stereoselective hydroxylation of 10-undecenoic acid with a light-driven P450 BM3 biocatalyst yielding a valuable synthon for natural product synthesis. , 2014, Bioorganic & medicinal chemistry.

[105]  Frances H Arnold,et al.  Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3. , 2003, Journal of the American Chemical Society.

[106]  Huimin Zhao,et al.  Inverting the enantioselectivity of P450pyr monooxygenase by directed evolution. , 2010, Chemical communications.

[107]  N. Vermeulen,et al.  A Single Active Site Mutation Inverts Stereoselectivity of 16‐Hydroxylation of Testosterone Catalyzed by Engineered Cytochrome P450 BM3 , 2012, Chembiochem : a European journal of chemical biology.

[108]  David Baker,et al.  A novel semi-biosynthetic route for artemisinin production using engineered substrate-promiscuous P450(BM3). , 2009, ACS chemical biology.

[109]  V. Urlacher,et al.  Regioselective hydroxylation of norisoprenoids by CYP109D1 from Sorangium cellulosum So ce56 , 2010, Applied Microbiology and Biotechnology.

[110]  A. Munro,et al.  Variations on a (t)heme--novel mechanisms, redox partners and catalytic functions in the cytochrome P450 superfamily. , 2007, Natural product reports.

[111]  I. Antes,et al.  Theoretical and Experimental Evaluation of a CYP106A2 Low Homology Model and Production of Mutants with Changed Activity and Selectivity of Hydroxylation , 2008, Chembiochem : a European journal of chemical biology.

[112]  Gheorghe-Doru Roiban,et al.  Achieving Regio‐ and Enantioselectivity of P450‐Catalyzed Oxidative CH Activation of Small Functionalized Molecules by Structure‐Guided Directed Evolution , 2012, Chembiochem : a European journal of chemical biology.

[113]  Andreas Vogel,et al.  Expanding the substrate scope of enzymes: combining mutations obtained by CASTing. , 2006, Chemistry.

[114]  Manfred T Reetz,et al.  A Method for High-Throughput Screening of Enantioselective Catalysts. , 1999, Angewandte Chemie.

[115]  T. Poulos Reversing enzyme specificity , 1989, Nature.

[116]  N. Turner,et al.  Cytochromes P450 as useful biocatalysts: addressing the limitations. , 2011, Chemical communications.

[117]  P. Gannett,et al.  The Use of Immobilized Cytochrome P4502C9 in PMMA-Based Plug Flow Bioreactors for the Production of Drug Metabolites , 2014, Applied Biochemistry and Biotechnology.

[118]  Manfred T Reetz,et al.  Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes , 2007, Nature Protocols.

[119]  Ulrich Schwaneberg,et al.  Regioselective o-hydroxylation of monosubstituted benzenes by P450 BM3. , 2013, Angewandte Chemie.

[120]  Nico P E Vermeulen,et al.  Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs. , 2014, Bioorganic & medicinal chemistry.

[121]  L. Wong,et al.  Cytochrome P450 monooxygenases. , 1998, Current opinion in chemical biology.

[122]  K. Drauz,et al.  Enzyme Catalysis in Organic Synthesis: DRAUZ:ENZYME CAT.3VLS.3ED O-BK , 2012 .

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

[124]  M. Honing,et al.  Role of residue 87 in substrate selectivity and regioselectivity of drug-metabolizing cytochrome P450 CYP102A1 M11 , 2011, JBIC Journal of Biological Inorganic Chemistry.

[125]  Davidr . Evans,et al.  Substrate-directable chemical reactions , 1993 .

[126]  Xing-Cong Li,et al.  New α-Tetralone Galloylglucosides from the Fresh Pericarps of Juglans sigillata. , 2010, Helvetica chimica acta.

[127]  T. Brück,et al.  Production of Macrocyclic Sesqui‐ and Diterpenes in Heterologous Microbial Hosts: A Systems Approach to Harness Nature’s Molecular Diversity , 2014 .

[128]  Son Quang Pham,et al.  Evolving P450pyr hydroxylase for highly enantioselective hydroxylation at non-activated carbon atom. , 2012, Chemical communications.

[129]  M. Sanford,et al.  Controlling site selectivity in palladium-catalyzed C-H bond functionalization. , 2012, Accounts of chemical research.

[130]  U. Schwaneberg,et al.  Benzylic hydroxylation of aromatic compounds by P450 BM3 , 2013 .

[131]  Andreas Schmid,et al.  Whole-cell biocatalysis for selective and productive C-O functional group introduction and modification. , 2013, Chemical Society reviews.

[132]  S. Bell,et al.  Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3. , 2005, Organic & biomolecular chemistry.

[133]  T. Ahn,et al.  Regioselective hydroxylation of 17β-estradiol by mutants of CYP102A1 from Bacillus megaterium , 2014, Biotechnology Letters.

[134]  Manfred T Reetz,et al.  Iterative saturation mutagenesis: a powerful approach to engineer proteins by systematically simulating Darwinian evolution. , 2014, Methods in molecular biology.

[135]  S. Imaoka,et al.  Construction and engineering of a thermostable self-sufficient cytochrome P450. , 2009, Biochemical and biophysical research communications.