Achieving Regio‐ and Enantioselectivity of P450‐Catalyzed Oxidative CH Activation of Small Functionalized Molecules by Structure‐Guided Directed Evolution

Directed evolution of the monooxygenase P450‐BM3 utilizing iterative saturation mutagenesis at and near the binding site enables a high degree of both regio‐ and enantioselectivity in the oxidative hydroxylation of cyclohexene‐1‐carboxylic acid methyl ester. Wild‐type P450‐BM3 is 84 % regioselective for the allylic 3‐position with 34 % enantioselectivity in favor of the R alcohol. Mutants enabling R selectivity (>95 % ee) or S selectivity (>95 % ee) were evolved, while reducing other oxidation products and thus maximizing regioselectivity to >93 %. Control of the substrate‐to‐enzyme ratio is necessary for obtaining optimal and reproducible enantioselectivities, an observation which is important in future protein engineering of these mono‐oxygenases. An E. coli strain capable of NADPH regeneration was also engineered, simplifying directed evolution of P450 enzymes in general. These synthetic results set the stage for subsequent stereoselective and stereospecific chemical transformations to form more complex compounds, thereby illustrating the viability of combining genetically altered enzymes as catalysts in organic chemistry with traditional chemical methods.

[1]  MilesCS ChapmanSK LysekDA MacKayAC ReidGA HanzlikRP MunroA NobleMA.  Roles of key active-site residues in flavocytochrome P450 BM3. , 1999 .

[2]  J. E. Thomson,et al.  Parallel kinetic resolution of tert-butyl (RS)-3-oxy-substituted cyclopent-1-ene-carboxylates for the asymmetric synthesis of 3-oxy-substituted cispentacin and transpentacin derivatives. , 2008, Organic & biomolecular chemistry.

[3]  Kentaro Miyazaki,et al.  Creating random mutagenesis libraries using megaprimer PCR of whole plasmid. , 2002, BioTechniques.

[4]  J. Takeuchi,et al.  Regioselective palladium-catalysed amination of 4-chloroacetoxyalk-2-enoic esters: synthesis of pyrrol-2(5H)-ones (4-but-2-enelactams) , 1987 .

[5]  Preparation of (R)- and (S)-N-protected 3-hydroxypyrrolidines by hydroxylation with Sphingomonas sp. HXN-200, a highly active, regio- and stereoselective, and easy to handle biocatalyst. , 2001, The Journal of organic chemistry.

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

[7]  J. Halpert,et al.  Plasticity of CYP2B enzymes: structural and solution biophysical methods. , 2012, Current drug metabolism.

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

[9]  K. Miyazaki.  MEGAWHOP cloning: a method of creating random mutagenesis libraries via megaprimer PCR of whole plasmids. , 2011, Methods in enzymology.

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

[11]  D. G. Gibson,et al.  Enzymatic Assembly of Overlapping DNA Fragments , 2011, Methods in Enzymology.

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

[13]  Manfred T Reetz,et al.  Addressing the Numbers Problem in Directed Evolution , 2008, Chembiochem : a European journal of chemical biology.

[14]  W. Atkins,et al.  Interactions of cytochrome P450s with their ligands. , 2011, Archives of biochemistry and biophysics.

[15]  Cathy H. Wu,et al.  UniProt: the Universal Protein knowledgebase , 2004, Nucleic Acids Res..

[16]  Jan Marienhagen,et al.  Advances in generating functional diversity for directed protein evolution. , 2009, Current opinion in chemical biology.

[17]  Vlada B Urlacher,et al.  Cytochrome P450 monooxygenases: perspectives for synthetic application. , 2006, Trends in biotechnology.

[18]  W. Adam,et al.  Oxidation of Organic Compounds by Dioxiranes: Adam/Oxidation of Organic Compounds by Dioxiranes , 2009 .

[19]  F. Arnold,et al.  Engineering Cytochrome P450 BM3 for Terminal Alkane Hydroxylation , 2006 .

[20]  Frances H Arnold,et al.  Cytochrome P450: taming a wild type enzyme. , 2011, Current opinion in biotechnology.

[21]  Stefan Lutz,et al.  Protein engineering handbook. , 2009 .

[22]  J. H. Rigby,et al.  Preparation of highly substituted 2-pyridones by reaction of vinyl isocyanates and enamines , 1989 .

[23]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[24]  N. Vermeulen,et al.  Application of drug metabolising mutants of cytochrome P450 BM3 (CYP102A1) as biocatalysts for the generation of reactive metabolites. , 2008, Chemico-biological interactions.

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

[26]  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.

[27]  Dan S. Tawfik,et al.  Advances in laboratory evolution of enzymes. , 2008, Current opinion in chemical biology.

[28]  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.

[29]  F. Guengerich,et al.  Measurement of cytochrome P450 and NADPH–cytochrome P450 reductase , 2009, Nature Protocols.

[30]  Chankyu Park,et al.  Conversion of Methylglyoxal to Acetol by Escherichia coli Aldo-Keto Reductases , 2005, Journal of bacteriology.

[31]  Phil S. Baran,et al.  Wenn C‐H‐Bindungen sprechen könnten – selektive Oxidationen von C‐H‐Bindungen , 2011 .

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

[33]  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.

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

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

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

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

[38]  M. Cryle,et al.  The Role of the Conserved Threonine in P450BM3 Oxygen Activation: Substrate‐Determined Hydroxylation Activity of the Thr268Ala Mutant , 2008, Chembiochem : a European journal of chemical biology.

[39]  S. Danishefsky,et al.  The use of trans-methyl .beta.-nitroacrylate in Diels-Alder reactions , 1978 .

[40]  Frances H Arnold,et al.  Enantioselective epoxidation of terminal alkenes to (R)- and (S)-epoxides by engineered cytochromes P450 BM-3. , 2006, Chemistry.

[41]  U. Schwaneberg,et al.  A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes. , 2001, Journal of biotechnology.

[42]  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.

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

[44]  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.

[45]  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.

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

[47]  E. Corey,et al.  A short enantioselective pathway for the synthesis of the anti-influenza neuramidase inhibitor oseltamivir from 1,3-butadiene and acrylic acid. , 2006, Journal of the American Chemical Society.

[48]  U. Baumann,et al.  An efficient one-step site-directed and site-saturation mutagenesis protocol. , 2004, Nucleic acids research.

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

[50]  T. Ost,et al.  Rational re‐design of the substrate binding site of flavocytochrome P450 BM3 , 2000, FEBS letters.

[51]  A. Warshel,et al.  Electrostatic basis for enzyme catalysis. , 2006, Chemical reviews.

[52]  H. Ruijssenaars,et al.  Testosterone 15beta-hydroxylation by solvent tolerant Pseudomonas putida S12. , 2007, Journal of biotechnology.

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

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

[55]  B. Trost,et al.  Allylic alkylation. Palladium-catalyzed substitutions of allylic carboxylates. Stereo- and regiochemistry , 1980 .

[56]  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.

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

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

[59]  R. Friesner,et al.  Thermal equilibrium of high- and low-spin forms of cytochrome P450 BM-3: repositioning of the substrate? , 2005, Journal of the American Chemical Society.

[60]  E. Joly,et al.  An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes. , 1998, Nucleic acids research.

[61]  C. Cativiela,et al.  Stereoselective synthesis of ?-amino acids , 2007 .

[62]  D. Gin,et al.  Asymmetric synthetic access to the hetisine alkaloids: total synthesis of (+)-nominine. , 2008, Chemistry.

[63]  S. Sligar,et al.  Spectroscopic features of cytochrome P450 reaction intermediates. , 2011, Archives of biochemistry and biophysics.

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

[65]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[66]  A. Warshel,et al.  Exploration of the cytochrome c oxidase pathway puzzle and examination of the origin of elusive mutational effects. , 2011, Biochimica et biophysica acta.

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

[68]  L. Que,et al.  Targeting Specific C-H Bonds for Oxidation , 2006, Science.

[69]  D. Hilvert,et al.  Protein design by directed evolution. , 2008, Annual review of biophysics.

[70]  R. Breslow,et al.  Metal coordination-directed hydroxylation of steroids with a novel artificial P-450 catalyst. , 2006, Organic letters.

[71]  J. Groves,et al.  Alkane-oxidizing metalloenzymes in the carbon cycle. , 2011, Metallomics : integrated biometal science.

[72]  Frank Buchholz,et al.  A new logic for DNA engineering using recombination in Escherichia coli , 1998, Nature Genetics.

[73]  J. Groves,et al.  A highly reactive p450 model compound I. , 2009, Journal of the American Chemical Society.

[74]  U. Schwaneberg,et al.  Directed evolution of oxygenases: screening systems, success stories and challenges. , 2007, Combinatorial chemistry & high throughput screening.

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

[76]  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.

[77]  B. Trost,et al.  Development of a concise synthesis of (-)-oseltamivir (Tamiflu). , 2011, Chemistry.

[78]  H. Hogrefe,et al.  Creating randomized amino acid libraries with the QuikChange Multi Site-Directed Mutagenesis Kit. , 2002, BioTechniques.

[79]  David W. Russell,et al.  The Condensed Protocols From Molecular Cloning: A Laboratory Manual , 2006 .

[80]  Vlada B Urlacher,et al.  Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. , 2012, Trends in biotechnology.

[81]  Yosephine Gumulya,et al.  Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates , 2008, Applied Microbiology and Biotechnology.

[82]  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.

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

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

[85]  M. T. Reetz.  Gerichtete Evolution stereoselektiver Enzyme: Eine ergiebige Katalysator-Quelle f r asymmetrische Reaktionen , 2011 .

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

[87]  Sabine Laschat,et al.  Rational Design of a Minimal and Highly Enriched CYP102A1 Mutant Library with Improved Regio‐, Stereo‐ and Chemoselectivity , 2009, Chembiochem : a European journal of chemical biology.

[88]  M. Toscano,et al.  Nanomolar Inhibition of Type II Dehydroquinase Based on the Enolate Reaction Mechanism , 2007, ChemMedChem.

[89]  V. Urlacher,et al.  Biotransformation of β-ionone by engineered cytochrome P450 BM-3 , 2006, Applied Microbiology and Biotechnology.

[90]  Keigo Kamata,et al.  Efficient stereo- and regioselective hydroxylation of alkanes catalysed by a bulky polyoxometalate , 2010, Nature Chemistry.

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

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

[93]  Manfred T Reetz,et al.  Directed evolution of enantioselective enzymes: iterative cycles of CASTing for probing protein-sequence space. , 2006, Angewandte Chemie.

[94]  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.

[95]  S. Silvestre,et al.  Catalytic oxidative processes in steroid chemistry: allylic oxidation, β-selective epoxidation, alcohol oxidations and remote functionalization reactions , 2006 .

[96]  S. de Vries,et al.  Spectroscopic characterization of cytochrome P450 Compound I. , 2011, Archives of biochemistry and biophysics.

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

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

[99]  M. Kanai,et al.  Second generation catalytic asymmetric synthesis of Tamiflu: allylic substitution route. , 2007, Organic letters.

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

[101]  M. White,et al.  A Predictably Selective Aliphatic C–H Oxidation Reaction for Complex Molecule Synthesis , 2007, Science.

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

[103]  J. Meinwald,et al.  Notes - The Ultraviolet Absorption Spectrum of 1,4-Cyclohexadien-1-carboxylic Acid, an Intermediate in the Preparation of 1,4-Cyclohexadienyl Methyl Ketone. , 1956 .

[104]  G. A. Berchtold,et al.  Aromatization of arene 1,2-oxides. Comparison of the aromatization pathways of 1-carboxy-, 1-carbomethoxy-, 1-formyl-, 1-(hydroxymethyl)-, and 1-(2-hydroxy-2-propyl)benzene oxide , 1981 .

[105]  G. Brudvig,et al.  Molecular Recognition in the Selective Oxygenation of Saturated C-H Bonds by a Dimanganese Catalyst , 2006, Science.

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

[107]  Mark A. Wall,et al.  Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution , 2007, Applied and Environmental Microbiology.

[108]  John M Woodley,et al.  Substrate Supply for Effective Biocatalysis , 2007, Biotechnology progress.

[109]  R D Schmid,et al.  P450 monooxygenase in biotechnology. I. Single-step, large-scale purification method for cytochrome P450 BM-3 by anion-exchange chromatography. , 1999, Journal of chromatography. A.

[110]  Manfred T Reetz,et al.  Constructing and Analyzing the Fitness Landscape of an Experimental Evolutionary Process , 2008, Chembiochem : a European journal of chemical biology.

[111]  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.

[112]  Sonia Rodriguez,et al.  Purification and Identification of an Escherichiacoli β‐Keto Ester Reductase as 2,5‐Diketo‐d‐gluconate Reductase YqhE , 2002, Biotechnology progress.