Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

Abstract

Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. P450s are considered as the most versatile biocatalysts in nature because of the vast variety of substrate structures and the types of reactions they catalyze. In particular, P450s can catalyze regio- and stereoselective oxidations of nonactivated C–H bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, bioremediation agents, flavors, and fragrances. Major efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. In this review, we focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals. We also discuss future opportunities for engineering and applications of the P450 systems. Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. P450s are considered as the most versatile biocatalysts in nature because of the vast variety of substrate structures and the types of reactions they catalyze. In particular, P450s can catalyze regio- and stereoselective oxidations of nonactivated C–H bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, bioremediation agents, flavors, and fragrances. Major efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. In this review, we focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals. We also discuss future opportunities for engineering and applications of the P450 systems. Cytochrome P450 enzymes (P450s) 3The abbreviations used are: P450 or CYPcytochrome P450FdRferredoxin reductaseFdxferredoxinAdxadrenodoxinAdRadrenodoxin reductaseCPRcytochrome P450 reductaseCpd 0I, and II, compound 0, I, and IIepPCRerror-prone polymerase chain reaction1α,25(OH)2D31α,25-dihydroxyvitamin D3PDBProtein Data BankSRSsubstrate recognition site. are a superfamily of heme-thiolate–containing proteins named for the characteristic state of the reduced, carbon monoxide (CO)-bound complex displaying a maximum UV-visible absorption band at 450 nm, due to the heme iron group being linked to the apoprotein via an axial conserved cysteine (1Guengerich F.P. Mechanisms of cytochrome P450-catalyzed oxidations.ACS Catal. 2018; 8 (31105987): 10964-1097610.1021/acscatal.8b03401Crossref PubMed Scopus (0) Google Scholar, 2Meunier B. de Visser S.P. Shaik S. Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes.Chem. Rev. 2004; 104 (15352783): 3947-398010.1021/cr020443gCrossref PubMed Scopus (1611) Google Scholar). cytochrome P450 ferredoxin reductase ferredoxin adrenodoxin adrenodoxin reductase cytochrome P450 reductase I, and II, compound 0, I, and II error-prone polymerase chain reaction 1α,25-dihydroxyvitamin D3 Protein Data Bank substrate recognition site. Since the first discovery of P450 as a pigment (the P denoting “pigment”) in rat liver microsomes in 1958 (3Klingenberg M. Pigments of rat liver microsomes.Arch. Biochem. Biophys. 1958; 75 (13534720): 376-38610.1016/0003-9861(58)90436-3Crossref PubMed Google Scholar), more than 370,000 P450 sequences have been released (UniProt), which are found in human, animals, plants, microbes, and even viruses, demonstrating their incredible and significant diversity in nature (4Nelson D.R. Cytochrome P450 diversity in the tree of life.Biochim. Biophys. Acta Proteins Proteom. 2018; 1866 (28502748): 141-15410.1016/j.bbapap.2017.05.003Crossref PubMed Scopus (0) Google Scholar). P450s play important roles in biosynthetic pathways for natural products, degradation of xenobiotics, biosynthesis of steroid hormones, and drug metabolism (5Zhang X. Li S. Expansion of chemical space for natural products by uncommon P450 reactions.Nat. Prod. Rep. 2017; 34 (28770915): 1061-108910.1039/C7NP00028FCrossref PubMed Google Scholar, 6Guengerich F.P. Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity.Chem. Res. Toxicol. 2001; 14 (11409933): 611-65010.1021/tx0002583Crossref PubMed Google Scholar). P450s are considered to be the most versatile biocatalysts in nature (7Coon M.J. Cytochrome P450: nature's most versatile biological catalyst.Annu. Rev. Pharmacol. Toxicol. 2005; 45 (15832443): 1-2510.1146/annurev.pharmtox.45.120403.100030Crossref PubMed Scopus (261) Google Scholar) and are involved in more than 20 different types of chemical oxidation reactions, including hydroxylation, epoxidation, decarboxylation, N- and O-dealkylation, nitration, and C–C bond coupling or cleavage, to name a few (5Zhang X. Li S. Expansion of chemical space for natural products by uncommon P450 reactions.Nat. Prod. Rep. 2017; 34 (28770915): 1061-108910.1039/C7NP00028FCrossref PubMed Google Scholar, 8Guengerich F.P. Munro A.W. Unusual cytochrome P450 enzymes and reactions.J. Biol. Chem. 2013; 288 (23632016): 17065-1707310.1074/jbc.R113.462275Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) (plus some reductions). Furthermore, the substrate diversity of P450s covers almost all classes of organic structures found in nature (e.g. terpenoids, polyketides, fatty acids, alkaloids, and polypeptides) (5Zhang X. Li S. Expansion of chemical space for natural products by uncommon P450 reactions.Nat. Prod. Rep. 2017; 34 (28770915): 1061-108910.1039/C7NP00028FCrossref PubMed Google Scholar, 9Podust L.M. Sherman D.H. Diversity of P450 enzymes in the biosynthesis of natural products.Nat. Prod. Rep. 2012; 29 (22820933): 1251-126610.1039/c2np20020aCrossref PubMed Scopus (156) Google Scholar, 10Rudolf J.D. Chang C.Y. Ma M. Shen B. Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function.Nat. Prod. Rep. 2017; 34 (28758170): 1141-117210.1039/C7NP00034KCrossref PubMed Google Scholar). The ubiquitous distribution and the multiplicity of reactions and substrates demonstrate the plasticity of P450 enzyme systems, providing a limitless space for mining, engineering, and designing P450 systems for practical catalysis. Among diverse functionalities, the most important is that P450s are capable of catalyzing the regio- and stereoselective oxidation of inert C–H bonds in complex molecular scaffolds under mild conditions, making them superior to many chemical catalysts and of great interest for pharmaceutical, chemical, and biotechnological applications. However, the narrow substrate scope of some P450s, low catalytic efficiency, low stability, dependence on redox partners, high cost of cofactors, and electron uncoupling have limited the industrial applications of P450s (11Sakaki T. Practical application of cytochrome P450.Biol. Pharm. Bull. 2012; 35 (22687473): 844-84910.1248/bpb.35.844Crossref PubMed Google Scholar, 12Bernhardt R. Urlacher V.B. Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations.Appl. Microbiol. Biotechnol. 2014; 98 (24848420): 6185-620310.1007/s00253-014-5767-7Crossref PubMed Scopus (201) Google Scholar). More recently, innovative P450 systems have been developed to fuel industrial projects with the use of a number of new engineering strategies (e.g. interactions of essential elements, including P450 itself, redox partner, substrate, and cofactor). These include the powerful directed evolution approach pioneered by the Nobel Laureate Frances H. Arnold, used to build unnatural but more robust P450 systems (13Arnold F.H. Design by directed evolution.Acc. Chem. Res. 1998; 31: 125-13110.1021/ar960017fCrossref Google Scholar). Several excellent reviews have covered the diversity, functions, novel chemistry, and applications of P450s (5Zhang X. Li S. Expansion of chemical space for natural products by uncommon P450 reactions.Nat. Prod. Rep. 2017; 34 (28770915): 1061-108910.1039/C7NP00028FCrossref PubMed Google Scholar, 10Rudolf J.D. Chang C.Y. Ma M. Shen B. Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function.Nat. Prod. 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For more insight into intriguing P450-related mechanisms and to deeply understand the strategies related to the practical application of P450 catalysis, we will focus on recent advances in P450 protein engineering, particularly engineering strategies for optimization of the interaction between P450s and redox partners. We will also consider substrate engineering, cofactor (NAD(P)H) regeneration, and several atypical strategies for engineering the electron transport system. Finally, a brief summary of P450-related metabolic engineering will be provided. In general, a P450 catalytic system includes four components: the substrate, a P450 enzyme for substrate binding and oxidative catalysis, the redox partner(s) that functions as an electron transfer shuttle, and the cofactor (NAD(P)H), which provides the reducing equivalents. Most P450s share a common sophisticated catalytic cycle (Fig. 1) (2Meunier B. de Visser S.P. Shaik S. Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes.Chem. Rev. 2004; 104 (15352783): 3947-398010.1021/cr020443gCrossref PubMed Scopus (1611) Google Scholar, 5Zhang X. Li S. Expansion of chemical space for natural products by uncommon P450 reactions.Nat. Prod. Rep. 2017; 34 (28770915): 1061-108910.1039/C7NP00028FCrossref PubMed Google Scholar, 18Jiang Y. Li S. Catalytic function and application of cytochrome P450 enzymes in biosynthesis and organic synthesis.Chinese J. Org. Chem. 2018; 38: 2307-232310.6023/cjoc201805055Crossref Scopus (4) Google Scholar), using the typical hydroxylation reaction as a paradigm, as shown in Fig. 1. The ferric resting state (generally) of a P450 (A) first accepts a substrate (RH), which displaces an active-site water molecule but does not bond directly to the iron. The ferric iron (FeIII) of the high-spin, substrate-bound complex (B) is then reduced to ferrous iron (FeII) (C) by one electron, transferred via a redox partner. Next, binding of dioxygen to FeII results in the [FeII O2] complex (D). The complex D is reduced by the second electron to form complex E, which uses a proton from solvent to generate a ferric hydroperoxo species [FeIII–OOH] (F), referred as to Compound 0 (Cpd 0). The O–O bond of Cpd 0 is cleaved upon the addition of the second proton and releases a molecule of water to generate the high-valent porphyrin π radical cation tetravalent iron [FeIV=O] (i.e. Compound I (Cpd I; G)). This highly reactive complex abstracts a hydrogen atom from the substrate, leading to the formation of the ferryl-hydroxo compound II (Cpd II; H). Subsequently, the hydroxylated product (R-OH) is formed by the reaction of the substrate radical with the hydroxyl group of Cpd II and released from the active site of complex I. Finally, a molecule of water returns to coordinate with FeIII, restoring the resting state A. The same catalytic cycle is initiated repeatedly as substrate molecules bind to the heme-centered active site of P450. It is worth noting that some P450s are capable of directly utilizing H2O2 as the sole electron and proton donor to form Cpd 0 and do catalysis via the so-called peroxide shunt pathway (Fig. 1, dashed arrows). However, this shunt pathway is greatly limited by the low efficiency and the low H2O2 tolerance of most P450s, except P450 peroxygenases (e.g. CYP152 subfamily) (19Matthews S. Belcher J.D. Tee K.L. Girvan H.M. McLean K.J. Rigby S.E. Levy C.W. Leys D. Parker D.A. Blankley R.T. Munro A.W. Catalytic determinants of alkene production by the cytochrome P450 peroxygenase OleTJE.J. Biol. Chem. 2017; 292 (28053093): 5128-514310.1074/jbc.M116.762336Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The well-studied and established catalytic cycle provides a theoretical basis and roadmap to understand and manipulate this P450 peroxygenase subfamily by protein and substrate engineering. Maintenance of the P450 catalytic cycle relies on continuous electron transport to the heme-iron by redox partners, which are complicated electron-transfer systems. Based on the types of redox partners and the P450-redox partner interaction relationships, P450 systems can be divided into five main classes (10Rudolf J.D. Chang C.Y. Ma M. Shen B. Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function.Nat. Prod. Rep. 2017; 34 (28758170): 1141-117210.1039/C7NP00034KCrossref PubMed Google Scholar, 11Sakaki T. Practical application of cytochrome P450.Biol. Pharm. Bull. 2012; 35 (22687473): 844-84910.1248/bpb.35.844Crossref PubMed Google Scholar, 15Xu L.H. Du Y.L. 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The Class II P450 system employed by eukaryotic organisms has a single-component redox partner, which is a membrane-bound protein containing both an FAD and an FMN domain, termed cytochrome P450 reductase (CPR). Class III P450 systems have a eukaryotic-like CPR naturally fused to the C terminus of the P450 domain through a flexible linker, represented by Bacillus megaterium P450BM3 (CYP102A1) (22Whitehouse C.J.C. Bell S.G. Wong L.-L. P450BM3(CYP102A1): connecting the dots.Chem. Soc. Rev. 2012; 41 (22008827): 1218-126010.1039/C1CS15192DCrossref PubMed Google Scholar). Class IV P450 systems are exemplified by P450 RhF from Rhodococcus sp. NCIMB 9784, whose FMN/Fe2S2-containing reductase domain forms a natural fusion with the P450 domain (23Roberts G.A. Celik A. Hunter D.J. Ost T.W. White J.H. Chapman S.K. Turner N.J. Flitsch S.L. A self-sufficient cytochrome P450 with a primary structural organization that includes a flavin domain and a [2Fe-2S] redox center.J. Biol. 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