Cytochrome P450 monooxygenases: an update on perspectives for synthetic application

Cytochrome P450 monooxygenases (P450s) are versatile biocatalysts that catalyze the regio- and stereospeci?c oxidation of non-activated hydrocarbons under mild conditions, which is a challenging task for chemical catalysts. Over the past decade impressive advances have been achieved via protein engineering with regard to activity, stability and specificity of P450s. In addition, a large pool of newly annotated P450s has attracted much attention as a source for novel biocatalysts for oxidation. In this review we give a short up-to-date overview of recent results on P450 engineering for technical applications including aspects of whole-cell biocatalysis with engineered recombinant enzymes. Furthermore, we focus on recently identified P450s with novel biotechnologically relevant properties.     

Extending the capabilities of nature's most versatile catalysts: directed evolution of mammalian xenobiotic-metabolizing P450s

Cytochrome P450 enzymes are amongst the most versatile enzymatic catalysts known. The ability to introduce a single atom of oxygen into an organic substrate has led to the diversification and exploitation of these enzymes throughout nature. Nowhere is this versatility more apparent than in the mammalian liver, where P450 monooxygenases catalyze the metabolic clearance of innumerate drugs and other environmental chemicals. In addition to the aromatic and aliphatic hydroxylations, N- and O-dealkylations, and heteroatom oxidations that are common in drug metabolism, many more unusual reactions catalyzed by P450s have been discovered, including reductions, group transfers and other biotransformations not typically associated with monooxygenases. A research area that shows great potential for development over the next few decades is the directed evolution of P450s as biocatalysts. Mammalian xenobiotic-metabolizing P450s are especially well suited to such protein engineering due to their ability to interact with relatively wide ranges of substrates with marked differences in structure and physicochemical properties. Typical characteristics, such as the low turnover rates and poor coupling seen during the metabolism of xenobiotics, as well as the enzyme specificity towards particular substrates and reactions, can be improved by directed evolution. This mini-review will cover the fundamental enabling technologies required to successfully engineer P450s, examine the work done to date on the directed evolution of mammalian forms, and provide a perspective on what will be required for the successful implementation of engineered enzymes.     

Recent advances in biocatalyst discovery,development and applications

Enzymes catalyze a wide range of biotransformations and have a great potential as environmentally friendly alternatives to classical chemical catalysts in various industrial applications. Recently, advanced techniques and strategies in enzyme discovery and engineering have led to the significant expansion of the quantity and functional diversity of biocatalysts, which has further allowed broader uses of biocatalysts in new processes, especially those traditionally enabled only by chemical catalysts. Here we highlight some of these recent advances with the focus on new approaches in biocatalyst discovery and development, and discuss new applications of selected biocatalysts including transaminases, cytochrome P450s, and Baeyer–Villiger monooxygenases.     

Progress towards the easier use of P450 enzymes

The cytochrome P450 enzymes (P450s or CYPs) form a large family of heme proteins involved in drug metabolism and in the biosynthesis of steroids, lipids, vitamins and natural products. Their remarkable ability to catalyze the insertion of oxygen into non-activated C-H bonds has attracted the interest of chemists for several decades. Very few chemical methods exist that directly hydroxylate aliphatic or aromatic C-H bonds, and most of them are not selective or of limited scope. Biocatalysts such as P450s represent a promising alternative: however, their applications have been limited by substrate specificity, low activity, poor stability and the need for cofactors. This review covers the attempts to overcome these limitations using approaches such as mutagenesis, chemical modifications, conditions engineering and immobilization.     

Cytochrome P450: taming a wild type enzyme

Protein engineering of cytochrome P450 monooxygenases (P450s) has been very successful in generating valuable non-natural activities and properties, allowing these powerful catalysts to be used for the synthesis of drug metabolites and in biosynthetic pathways for the production of precursors of artemisinin and paclitaxel. Collected experience indicates that the P450s are highly 'evolvable' - they are particularly robust to mutation in their active sites and readily accept new substrates and exhibit new selectivities. Their ability to adapt to new challenges upon mutation may reflect the nonpolar nature of their active sites as well as their high degree of conformational variability.     

New Trends in Cytochrome P450 Research at the Half-Century Mark

Cytochrome P450 enzymes have major roles in the metabolism of steroids, drugs, carcinogens, eicosanoids, and numerous other chemicals. The P450s are collectively considered the most diverse catalysts known in biochemistry, although they operate from a basic structural fold and catalytic mechanism. The four minireviews in this thematic series deal with the unusual aspects of catalytic reactions and electron transfer pathway organization, the structural diversity of P450s, and the expanding roles of P450s in disease and medicine.     

细胞色素P450 1A1和1B1靶向抗肿瘤前药研究进展

细胞色素P450（CYP）能催化各种内源性及外源性化合物的代谢,与多种肿瘤发生有关。其中CYP1A1参与多种前致癌物和致突变物的代谢活化,CYP1B1被认为在许多人癌细胞中特异性表达,参与药物的氧化代谢和前药的活化。CYP1A1和181已成为靶向抗肿瘤前药研究的新靶点。相继有大量相关研究报道,本文就近年来文献报道的CYP1A1和1B1靶向抗肿瘤前药研究进展。     

Cytochromes P450: exploiting diversity and enabling application as biocatalysts

The remarkable chemical reactivity and substrate range displayed by cytochromes P450 (P450s) renders them attractive as potential catalysts for a host of challenging chemical reactions in industry. The opportunities afforded by these biocatalysts are increased by the availability of greater diversity provided by the genomic resource and the variant libraries of well-known P450s produced by rational and random engineering techniques. The exploitation of this enormous diversity will require novel tools in screening, to identify enzyme reactions of interest, and also in the enabling of these valuable activities through protein engineering and bioprocess optimisation.     

Plant cytochrome P450-mediated herbicide metabolism

In the last two decades it has become apparent that enzymes of the P450 monooxygenase (P450) superfamily are responsible for the Phase I metabolism of numerous herbicides representing several classes of organic compounds. The majority of experimental evidence for P450 involvement in herbicide metabolism has been derived from in vitro studies in which the catalytic activity of plant microsomes towards herbicidal substrates was measured in the presence of various P450 inhibitors and activators. While the studies with microsomes elicited much appreciation for the pivotal roles of plant P450s in herbicide metabolism, detailed characterization of these enzymes only became possible after the isolation of genes encoding specific isoforms responsible for herbicide conversion. Several lines of evidence suggest that the development of herbicide resistance in weeds by enhanced detoxification is frequently associated with elevated levels of P450 activity. Enhanced detoxification-based herbicide resistance is particularly difficult to control, because it can involve resistance to multiple, chemically unrelated classes of herbicides. Continued research efforts are aimed at elucidating the role of P450s in the metabolic fates of herbicides in plants and the development of herbicide resistance in weeds. Recent advances made in the isolation and genetic manipulation of P450 enzymes have created new opportunities for their application in engineering herbicide tolerance and bioremediation.     

细胞色素P450酶系与除草剂代谢

细胞色素P450是广泛存在于动物、植物和微生物体内的一类具有混合功能的血红素氧化酶系。它不但能够催化苯丙烷类、萜类化合物和脂肪酸等内源性物质的生物合成 ,而且参与许多外源性物质包括除草剂等的生物氧化。综述了代谢除草剂的细菌、哺乳动物和植物细胞色素P450酶系 ,概述了细胞色素P450酶系参与除草剂代谢的作用方式 :脱烷基化作用、环甲基化羟基化作用和芳环的羟基化作用等。这些细胞色素P450酶系在培育除草剂抗性作物、生物安全和生物修复方面表现出了巨大的潜能     

Cytochrome P450 monooxygenases: perspectives for synthetic application

Cytochrome P450 monooxygenases are versatile biocatalysts that introduce oxygen into a vast range of molecules. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds. However, the potential of P450 monooxygenases has not been fully exploited; there are some drawbacks limiting the broader implementation of these catalysts for commercial needs. Protein engineering has produced P450 enzymes with widely altered substrate specificities, substantially increased activity and higher stability. Furthermore, electrochemical and enzymatic approaches for the replacement or regeneration of NAD(P)H have been developed, enabling the more cost-effective use of P450 enzymes. In this review, we focus on the aspects relevant to the synthetic applications of P450 enzymes and their optimization for commercial needs.     

过氧化氢驱动的细胞色素P450酶研究进展

细胞色素P450酶在自然界中广泛存在,能催化多种类型的氧化反应,在有机合成和生物化工方面具有重要的应用潜力。尽管大多数P450酶通常需要辅酶和复杂的电子传递体系协助活化氧分子,一些P450酶也可以利用过氧化氢作为末端氧化剂,这极大地简化了催化循环,为P450酶的合成应用提供了一条新的简便途径。本文系统地介绍了几类过氧化氢驱动的P450酶催化体系,包括脂肪酸羟化酶P450SPα和P450BSβ、脂肪酸脱羧酶P450OleTJE、人工改造的羟化酶P450BM3和P450cam突变体、以及基于底物误识别策略的P450-H2O2体系。通过分析催化反应机制,本文探讨了P450-H2O2催化体系在目前存在的挑战和可能的解决途径,并对其进一步应用前景进行了展望。     

Cytochromes P450 and drug discovery

Cytochromes P450 (CYP) are a superfamily of haem-containing proteins distributed widely throughout nature. Historically, they have a central role in drug metabolism and following the advent of genomics they have been shown to have key roles in the biosynthesis of natural products which are used as medicines. Herein, we provide an overview of CYP systems with particular emphasis on their role as drug targets, their involvement in drug biosynthesis and potential strategies for developing new derivatives of drugs based on CYP engineering. The applied importance of CYPs for medicinal and biotechnological applications will also be discussed.     

Prediction of binding modes for ligands in the cytochromes P450 and other heme-containing proteins

The cytochromes P450 (P450s) are a family of heme-containing monooxygenase enzymes involved in a variety of functions, including the metabolism of endogenous and exogenous substances in the human body. During lead optimization, and in drug development, many potential drug candidates are rejected because of the affinity they display for drug-metabolising P450s. Recently, crystal structures of human enzymes involved in drug metabolism have been determined, significantly augmenting the prospect of using structure-based design to modulate the binding and metabolizing properties of compounds against P450 proteins. An important step in the application of structure-based metabolic optimization is the accurate prediction of docking modes in heme binding proteins. In this paper we assess the performance of the docking program GOLD at predicting the binding mode of 45 heme-containing complexes. We achieved success rates of 64% and 57% for Chemscore and Goldscore respectively; these success rates are significantly lower than the value of 79% observed with both scoring functions for the full GOLD validation set. Re-parameterization of metal-acceptor interactions and lipophilicity of planar nitrogen atoms in the scoring functions resulted in a significant increase in the percentage of successful dockings against the heme binding proteins (Chemscore 73%, Goldscore 65%). The modified scoring functions will be useful in docking applications on P450 enzymes and other heme binding proteins.     

Marine invertebrate cytochrome P450: emerging insights from vertebrate and insects analogies

Cytochrome P450 enzymes (P450s) are responsible for the oxidative metabolism of a plethora of endogenous and exogenous substrates. P450s and associated activities have been demonstrated in numerous marine invertebrates belonging to the phyla Cnidaria, Annelida (Polychaeta), Mollusca, Arthropoda (Crustacea) and Echinodermata. P450s of marine invertebrates and vertebrates show considerable sequence divergence and the few orthologs reveal the selective constraint on physiologically significant enzymes. P450s are present in virtually all tissues of marine invertebrates, although high levels usually are found in hepatic-like organs and steroidogenic tissues. High-throughput technologies result in the rapid acquisition of new marine invertebrate P450 sequences; however, the understanding of their function is poor. Based on analogy to vertebrates and insects, it is likely that P450s play a pivotal role in the physiology of marine invertebrates by catalyzing the biosynthesis of signal molecules including steroids such as 20-hydroxyecdysone (the molting hormone of crustaceans). The metabolism of many exogenous compounds including benzo(a)pyrene (BaP), pyrene, ethoxyresorufin, ethoxycoumarin and aniline is mediated by P450 enzymes in tissues of marine invertebrates. P450 gene expression, protein levels and P450 mediated metabolism of xenobiotics are induced by PAHs in some marine invertebrate species. Thus, regulation of P450 enzyme activity may play a central role in the adaptation of animals to environmental pollutants. Emphasis should be put on the elucidation of the function and regulation of the ever-increasing number of marine invertebrate P450s.     

Unsaturated fatty acid regulation of cytochrome P450 expression via a CAR-dependent pathway

The liver is responsible for key metabolic functions, including control of normal homoeostasis in response to diet and xenobiotic metabolism/detoxification. We have shown previously that inactivation of the hepatic cytochrome P450 system through conditional deletion of POR (P450 oxidoreductase) induces hepatic steatosis, liver growth and P450 expression. We have exploited a new conditional model of POR deletion to investigate the mechanism underlying these changes. We demonstrate that P450 induction, liver growth and hepatic triacylglycerol (triglyceride) homoeostasis are intimately linked and provide evidence that the observed phenotypes result from hepatic accumulation of unsaturated fatty acids, which mediate these phenotypes by activation of the nuclear receptor CAR (constitutive androstane receptor) and, to a lesser degree, PXR (pregnane X receptor). To our knowledge this is the first direct evidence that P450s play a major role in controlling unsaturated fatty acid homoeostasis via CAR. The regulation of P450s involved in xenobiotic metabolism by this mechanism has potentially significant implications for individual responses to drugs and environmental chemicals.     

Determinants of thermostability in the cytochrome P450 fold

Cytochromes P450 are found throughout the biosphere in a wide range of environments, serving a multitude of physiological functions. The ubiquity of the P450 fold suggests that it has been co-opted by evolution many times, and likely presents a useful compromise between structural stability and conformational flexibility. The diversity of substrates metabolized and reactions catalyzed by P450s makes them attractive starting materials for use as biocatalysts of commercially useful reactions. However, process conditions impose different requirements on enzymes to those in which they have evolved naturally. Most natural environments are relatively mild, and therefore most P450s have not been selected in Nature for the ability to withstand temperatures above ~ 40 °C, yet industrial processes frequently require extended incubations at much higher temperatures. Thus, there has been considerable interest and effort invested in finding or engineering thermostable P450 systems. Numerous P450s have now been identified in thermophilic organisms and analysis of their structures provides information as to mechanisms by which the P450 fold can be stabilized. In addition, protein engineering, particularly by directed or artificial evolution, has revealed mutations that serve to stabilize particular mesophilic enzymes of interest. Here we review the current understanding of thermostability as it applies to the P450 fold, gleaned from the analysis of P450s characterized from thermophilic organisms and the parallel engineering of mesophilic forms for greater thermostability. We then present a perspective on how this information might be used to design stable P450 enzymes for industrial application. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

Adrenodoxin--a versatile ferredoxin

Mammalian adrenodoxin (Adx) has been known for many years as an essential electron mediator in mitochondrial cytochrome P450 systems. Because of its ability to support several cytochrome P450 enzymes, it is involved not only in adrenal steroid hormone biosynthesis but also in vitamin D and bile acid metabolism. Recently, Adx is increasingly gaining attention because of its potential for pharmaceutical industry and biotechnology. With human cytochromes P450 becoming important drug targets, suitable Adx-based screening systems have to be developed to test putative new drugs. Moreover, in artificial systems, Adx has been shown to functionally interact with diverse bacterial cytochromes P450 catalyzing a variety of chemically interesting reactions. Putative biotechnological applications of such Adx-containing reconstituted systems are discussed.     

Mechanism of the plant cytochrome P450 for herbicide resistance: a modelling study

Plant cytochrome P450 is a key enzyme responsible for the herbicide resistance but the molecular basis of the mechanism is unclear. To understand this, four typical plant P450s and a widely resistant herbicide chlortoluron were analysed by carrying out homology modelling, molecular docking, molecular dynamics simulations and binding free energy analysis. Our results demonstrate that: (i) the putative hydrophobic residues located in the F-helix and polar residues in I-helix are critical in the herbicide resistance; (ii) the binding mode analysis and binding free energy calculation indicate that the distance between catalytic site of chlortoluron and heme of P450, as well as the binding affinity are key elements affecting the resistance for plants. In conclusion, this work provides a new insight into the interactions of plant P450s with herbicide from a molecular level, offering valuable information for the future design of novel effective herbicides which also escape from the P450 metabolism.     

Genome-wide analysis of cytochrome P450 monooxygenase genes in the silkworm, Bombyx mori

Based on the advances in the silkworm genome project, a new genome-wide analysis of cytochrome P450 genes was performed. A total of 84 CYP-related sequences were identified and could be classified into 26 families and 47 subfamilies according to standard nomenclature. Seventy eight of the eighty four genes appear to be functional and six are probable pseudogenes. The distribution of Bombyx mori P450s in the genome shows that most of them are tandem arranged on chromosomes, only 34 genes are present as singletons, with 8 clusters including 3 or more than 3 genes. Sequence alignments were used to reconstruct phylogenetic trees and to analyze the intron-exon organizations of the functional genes. The conserved intron positioning agrees perfectly with their common grouping on the tree. The presence of three extremely ancient introns which are conserved across different clans indicates that a few introns are still highly conserved after they have undergone extensive evolutionary changes of B. mori P450 duplication and divergence. Comparison of the P450s from B. mori to the P450s from Drosophila melanogaster shows that the expansion is not uniform across the gene families. Remarkably, two mitochondrial families, the B. mori CYP333 and D. melanogaster Cyp12, formed two orthologous groups in the phylogenetic tree. All CYP333s can be proposed to be related to xenobiotic metabolism in accordance with the D. melanogaster Cyp12s. The characterization and evolutionary analysis of P450s from B. mori in the current study provide useful information for understanding the characteristics and diversity of P450s from B. mori and the baseline for functional analyses of individual P450s in this model Lepidopteran insect.