The role of the conserved threonine in P450 BM3 oxygen activation: substrate-determined hydroxylation activity of the Thr268Ala mutant

The hydroxylation activity of the Thr268Ala mutant of P450(BM3) has been shown to occur to varying degrees with small alterations in the structure of a fatty-acid substrate. Ten substrates were investigated, including straight chain, branched chain and cis-cyclopropyl substituted fatty acids with a straight-chain length that varied between 12 and 16 carbon atoms. The efficacy of the hydroxylation activity appeared to be governed by the chain length of the substrate. Substrates possessing 14 to 15 carbons afforded the highest levels of activity, which were comparable with the wild-type enzyme. Outside of this window, straight-chain fatty acids showed reduced activity over the other substrate types. These results provide a cautionary tale concerning the loss of ferryl activity in such cytochrome P450 threonine to alanine mutants, as the nature of the substrate can determine the extent to which hydroxylation chemistry is abolished.     

A Promiscuous Bacterial P450: The Unparalleled Diversity of BM3 in Pharmaceutical Metabolism

CYP102A1 (BM3) is a catalytically self-sufficient flavocytochrome fusion protein isolated from Bacillus megaterium, which displays similar metabolic capabilities to many drug-metabolizing human P450 isoforms. BM3′s high catalytic efficiency, ease of production and malleable active site makes the enzyme a desirable tool in the production of small molecule metabolites, especially for compounds that exhibit drug-like chemical properties. The engineering of select key residues within the BM3 active site vastly expands the catalytic repertoire, generating variants which can perform a range of modifications. This provides an attractive alternative route to the production of valuable compounds that are often laborious to synthesize via traditional organic means. Extensive studies have been conducted with the aim of engineering BM3 to expand metabolite production towards a comprehensive range of drug-like compounds, with many key examples found both in the literature and in the wider industrial bioproduction setting of desirable oxy-metabolite production by both wild-type BM3 and related variants. This review covers the past and current research on the engineering of BM3 to produce drug metabolites and highlights its crucial role in the future of biosynthetic pharmaceutical production.     

细胞色素P450 BM-3羟基化吲哚能力的半理性改造

为进一步改造细胞色素P450 BM-3酶对吲哚的羟基化能力,以P450 BM-3结构与功能关系的推测为指导,选择突变酶P450 BM-3 （A74G/F87V/L188Q/E435T）为父本,在可能影响P450 BM-3催化吲哚羟基化区域选择性的D168位点进行定点饱和突变,根据全细胞催化产物颜色及组成进行筛选,得到了产物组成、酶动力学性质与父本不同的两个突变酶。突变酶D168W的吲哚羟基化产物中90%是靛玉红,而另一个突变酶D168R的产物中87%是靛蓝,产物组成均不同于亲本。在催化吲哚羟基化时,D168W的k_cat与父本相当,但K_m却是父本的4.8倍,催化活力只有父本的20%;而D168R的k_cat是父本的1.9倍,K_m是父本的82%,催化活力比父本提高了1.37倍。结果表明,在E435T突变上叠加D168位氨基酸残基突变对酶的催化性质产生了单一位点突变所不具有的协同效应,对酶催化的区域选择性和催化活力都有显著影响,以致改变了催化产物组成。这种基于知识的半理性定向进化方法由于是在关键位点进行突变,因此突变目的性强、突变效果显著。     

The FMN “140s Loop” of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450

Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the “140s” FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the “140s loop” by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the “140s loop”.     

Revisiting Cytochrome P450‐Mediated Oxyfunctionalization of Linear and Cyclic Alkanes

Cytochrome P450 monooxygenases (CYPs) of the CYP153 family catalyse terminal hydroxylation of n‐alkanes. Alkane hydroxylating mutants of self‐sufficient CYP102A1 have also been described. We evaluated two CYP153s (a three‐component system and a fused self‐sufficient CYP), wild‐type CYP102A1 and nine CYP102A1 mutants, for the conversion of three cycloalkanes (C₆, C₇ and C₈) and three n‐alkanes (C₆, C₈ and C₁₀) using whole cells (WCs) and crude cell‐free extracts (CFEs). The aim was to identify substrate–enzyme combinations that give high product titres and space‐time yields (STYs). Comparisons were made using total turnover numbers (TTNs) and turnover frequencies (TOFs) to normalize for CYP expression. Reactions were carried out using high enzyme and substrate concentrations compatible with high STYs. Under these conditions CYP102A1 and the double R47L,Y51F mutant, although not regioselective, performed better on all substrates in terms of product titres over 8 h, and thus STYs and TTNs, than heavily mutated variants that have been reported to give very high TOFs. CYP153A6, with its ferredoxin (Fdx) and ferredoxin reductase (FdR), emerged as the superior catalyst for conversion of n‐alkanes. In addition to its excellent regioselectivity it also gave the highest final product titres and STYs in WC conversions of hexane and octane. Interaction with FdR and Fdx initially limited performance in CFEs, but with additional FdR and Fdx gave 1‐octanol titres of 50 mmol⋅L_BRM⁻¹ and TTNs exceeding 12,000 over 18 h, rivalling results reported with self‐sufficient CYPs. Selecting biocatalysts for application requires caution, since experimental conditions such as amount of substrate added and solubility as well as cofactor dependence and regeneration can have a profound effect on catalyst performance, while stability and efficiency with regard to cofactor usage (coupling efficiency) are at least as important as TOFs when high product titres and STYs are the target.

     

Engineering Cytochrome P450 BM3 for Terminal Alkane Hydroxylation

Enzymes that catalyze the terminal hydroxylation of alkanes could be used to produce more valuable chemicals from hydrocarbons. Cytochrome P450 BM3 from Bacillus megaterium hydroxylates medium‐chain fatty acids at subterminal positions at high rates. To engineer BM3 for terminal alkane hydroxylation, we performed saturation mutagenesis at selected active‐site residues of a BM3 variant that hydroxylates alkanes. Recombination of beneficial mutations generated a library of BM3 mutants that hydroxylate linear alkanes with a wide range of regioselectivities. Mutant 77‐9H exhibits 52% selectivity for the terminal position of octane. This regioselectivity is octane‐specific and does not transfer to other substrates, including shorter and longer hydrocarbons or fatty acids. These results show that BM3 can be readily molded for regioselective oxidation.     

定点突变提高细胞色素P450 BM-3吲哚羟基化能力

为进一步提高细胞色素P450 BM-3(A74G/F87V/L188Q)对吲哚的羟基化能力,根据酶结构与功能的关系,以突变酶E435T为基础,在168位点引入D168L突变,获得了吲哚羟基化能力得到显著提高的突变酶D168L/E435T。突变酶对吲哚的K_m为1.72 mmol·L^-1(父本2.09 mmol·L^-1),转化数(k_cat)为28.15 min^-1(父本4.04 min^-1),表明D168L定点突变可以略微提高酶对底物的亲和力,但主要的效应是促进了底物的转化速率,这两个效应的综合表现是使酶的催化效率(k_cat K_m^-1)比父本酶提高了8.48倍。此外,产物中副产物靛玉红的比例也降低为1.2%(父本7.3%),这说明该突变酶催化吲哚的区域选择性上也更有利于靛蓝的生成。     

Regioselectivity and Activity of Cytochrome P450 BM‐3 and Mutant F87A in Reactions Driven by Hydrogen Peroxide

Cytochrome P450 BM‐3 (EC 1.14.14.1) is a monooxygenase that utilizes NADPH and dioxygen to hydroxylate fatty acids at subterminal positions. The enzyme is also capable of functioning as a peroxygenase in the same reaction, by utilizing hydrogen peroxide in place of the reductase domain, cofactor and oxygen. As a starting point for developing a practically useful hydroxylation biocatalyst, we compare the activity and regioselectivity of wild‐type P450 BM‐3 and its F87A mutant on various fatty acids. Neither enzyme catalyzes terminal hydroxylation under any of the conditions studied. While significantly enhancing peroxygenase activity, the F87A mutation also shifts hydroxylation further away from the terminal position. The H₂O₂‐driven reactions with either the full‐length BM‐3 enzyme or the heme domain are slow, but yield product distributions very similar to those generated when using NADPH and O₂.     

Structural Diversification of Macrolactones by Substrate‐Flexible Cytochrome P450 Monooxygenases

The substrate flexibilities of several cytochrome P450 monooxygenases involved in macrolide biosynthesis were investigated to test their potential for the generation of novel macrolides. PikC hydroxylase in the pikromycin producer Streptomyces venezuelae accepted oleandomycin as an alternative substrate and introduced a hydroxy group at the C‐4 position, which is different from the intrinsic C‐12 hydroxylation position in the natural substrate. This is the first report of C‐4 hydroxylation activity of cytochrome P450 monooxygenase involved in the biosynthesis of 14‐membered macrolides. EryF hydroxylase from the erythromycin biosynthetic pathway of Saccharopolyspora erythraea and OleP oxidase from the oleandomycin biosynthetic pathway of Streptomyces antibioticus also showed a certain degree of plasticity towards alternative substrates. In particular, EryF and OleP were found to oxidize a 12‐membered macrolactone as an alternative substrate. These results demonstrate the potential usefulness of these enzymes to diversify macrolactones by post‐PKS oxidations.     

A Diversified Library of Bacterial and Fungal Bifunctional Cytochrome P450 Enzymes for Drug Metabolite Synthesis

Innovative biohydroxylation catalysts for the preparation of drug metabolites were developed from scratch. A set of bacterial and fungal sequences of putative and already known bifunctional P450 enzymes was identified by protein sequence alignments, expressed in Escherichia coli and characterised. Notably, a fungal self‐sufficient cytochrome P450 (CYP) from Aspergillus fumigatus turned out to be especially stable during catalyst preparation and application and also in presence of organic co‐solvents. To enhance the catalytic activity and broaden the substrate specificity of those variants with high expression levels prominent single mutations were introduced. Selected improved variants were then used as lyophilised bacterial lysates for the synthesis of 4′‐hydroxydiclofenac and 6‐hydroxychlorzoxazone, the two metabolites of active pharmaceutical compounds diclofenac and chlorzoxazone representing the same metabolites as generated by human P450s.     

Co-Crystal Structure-Guided Optimization of Dual-Functional Small Molecules for Improving the Peroxygenase Activity of Cytochrome P450BM3

We recently developed an artificial P450–H₂O₂ system assisted by dual-functional small molecules (DFSMs) to modify the P450BM3 monooxygenase into its peroxygenase mode, which could be widely used for the oxidation of non-native substrates. Aiming to further improve the DFSM-facilitated P450–H₂O₂ system, a series of novel DFSMs having various unnatural amino acid groups was designed and synthesized, based on the co-crystal structure of P450BM3 and a typical DFSM, N-(ω-imidazolyl)-hexanoyl-L-phenylalanine, in this study. The size and hydrophobicity of the amino acid residue in the DFSM drastically affected the catalytic activity (up to 5-fold), stereoselectivity, and regioselectivity of the epoxidation and hydroxylation reactions. Docking simulations illustrated that the differential catalytic ability among the DFSMs is closely related to the binding affinity and the distance between the catalytic group and heme iron. This study not only enriches the DFSM toolbox to provide more options for utilizing the peroxide-shunt pathway of cytochrome P450BM3, but also sheds light on the great potential of the DFSM-driven P450 peroxygenase system in catalytic applications based on DFSM tunability.     

Riboflavin Is Directly Involved in N-Dealkylation Catalyzed by Bacterial Cytochrome P450 Monooxygenases

Like a vast number of enzymes in nature, bacterial cytochrome P450 monooxygenases require an activated form of flavin as a cofactor for catalytic activity. Riboflavin is the precursor of FAD and FMN that serves as indispensable cofactor for flavoenzymes. In contrast to previous notions, herein we describe the identification of an electron-transfer process that is directly mediated by riboflavin for N-dealkylation by bacterial P450 monooxygenases. The electron relay from NADPH to riboflavin and then via activated oxygen to heme was proposed based on a combination of X-ray crystallography, molecular modeling and molecular dynamics simulation, site-directed mutagenesis and biochemical analysis of representative bacterial P450 monooxygenases. This study provides new insights into the electron transfer mechanism in bacterial P450 enzyme catalysis and likely in yeasts, fungi, plants and mammals.     

Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.     

Structural Analysis of Cytochrome P450 105N1 Involved in the Biosynthesis of the Zincophore, Coelibactin

Coelibactin is a putative non-ribosomally synthesized peptide with predicted zincophore activity and which has been implicated in antibiotic regulation in Streptomyces coelicolor A3(2). The coelibactin biosynthetic pathway contains a stereo- and regio-specific monooxygenation step catalyzed by a cytochrome P450 enzyme (CYP105N1). We have determined the X-ray crystal structure of CYP105N1 at 2.9 Å and analyzed it in the context of the bacterial CYP105 family as a whole. The crystal structure reveals a channel between the α-helical domain and the β-sheet domain exposing the heme pocket and the long helix I to the solvent. This wide-open conformation of CYP105N1 may be related to the bulky substrate coelibactin. The ligand-free CYP105N1 structure has enough room in the substrate access channel to allow the coelibactin to enter into the active site. Analysis of typical siderophore ligands suggests that CYP105N1 may produce derivatives of coelibactin, which would then be able to chelate the zinc divalent cation.     

响应面法优化工程菌产细胞色素P450 BM-3的发酵条件

采用快速有效的数学统计方法对工程菌产细胞色素P450 BM-3的发酵条件进行了优化.首先利用Plackett-Burman设计从众多影响产P450 BM-3的因素中筛选出影响较大的4个因素：FeCl₃含量、诱导时机、诱导时间和接种量.在此基础上再利用响应面法中的杂合设计进行优化,通过拟合得到响应曲面函数,获得了最佳的实验条件.在该实验条件下,P450 BM-3酶活从37.6×10^-3U•ml^-1提高到57.9×10^-3U•ml^-1.     

Mammalian Cytochrome P450-Dependent Metabolism of Polychlorinated Dibenzo-p-dioxins and Coplanar Polychlorinated Biphenyls

Polychlorinated dibenzo-p-dioxins (PCDDs) and coplanar polychlorinated biphenyls (PCBs) contribute to dioxin toxicity in humans and wildlife after bioaccumulation through the food chain from the environment. The authors examined human and rat cytochrome P450 (CYP)-dependent metabolism of PCDDs and PCBs. A number of human CYP isoforms belonging to the CYP1 and CYP2 families showed remarkable activities toward low-chlorinated PCDDs. In particular, human CYP1A1, CYP1A2, and CYP1B1 showed high activities toward monoCDDs, diCDDs, and triCDDs but no detectable activity toward 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-tetraCDD). Large amino acids located at putative substrate-recognition sites and the F-G loop in rat CYP1A1 contributed to the successful metabolism of 2,3,7,8-tetraCDD. Rat, but not human, CYP1A1 metabolized 3,3'',4,4'',5-pentachlorobiphenyl (CB126) to two hydroxylated metabolites. These metabolites are probably less toxic than is CB126, due to their higher solubility. Homology models of human and rat CYP1A1s and CB126 docking studies indicated that two amino acid differences in the CB126-binding cavity were important for CB126 metabolism. In this review, the importance of CYPs in the metabolism of dioxins and PCBs in mammals and the species-based differences between humans and rats are described. In addition, the authors reveal the molecular mechanism behind the binding modes of dioxins and PCBs in the heme pocket of CYPs.     

Bringing out the Potential of Wild-type Cytochrome P450s using Decoy Molecules: Oxygenation of Nonnative Substrates by Bacterial Cytochrome P450s

To bring out the potential of wild-type cytochrome P450s, we have developed a series of “decoy molecules” to change their high substrate specificity without any mutagenesis. Decoy molecules are inert dummy substrates with structures that are very similar to those of natural substrates. The decoy molecules force long-alkyl-chain fatty acid hydroxylases (P450_BSβ, P450_SPα, and P450BM3) to generate the active species and to catalyze oxidation of various substrates other than fatty acids. Interestingly, the catalytic activity was highly dependent on the structure of decoy molecules. Furthermore, the enantioselectivity of reactions catalyzed by P450_BSβ and P450_SPα was also dependent on the structure of decoy molecules. The decoy molecule system allows us to control reactions catalyzed by wild-type enzymes by designing decoy molecules.     

Hippocampal Expression of Cytochrome P450 1B1 in Penetrating Traumatic Brain Injury

Hippocampal dysfunction contributes to multiple traumatic brain injury sequala. Female rodents’ outcome is superior to male which has been ascribed the neuroprotective sex hormones 17β-estradiol and progesterone. Cytochrome P450 1B1 (CYP1B1) is an oxidative enzyme influencing the neuroinflammatory response by creating inflammatory mediators and metabolizing neuroprotective 17β-estradiol and progesterone. In this study, we aimed to describe hippocampal CYP1B1 mRNA expression, protein presence of CYP1B1 and its key redox partner Cytochrome P450 reductase (CPR) in both sexes, as well as the effect of penetrating traumatic brain injury (pTBI). A total 64 adult Sprague Dawley rats divided by sex received pTBI or sham-surgery and were assigned survival times of 1-, 3-, 5- or 7 days. CYP1B1 mRNA was quantified using in-situ hybridization and immunohistochemistry performed to verify protein colocalization. CYP1B1 mRNA expression was present in all subregions but greatest in CA2 irrespective of sex, survival time or intervention. At 3-, 5- and 7 days post-injury, expression in CA2 was reduced in male rats subjected to pTBI compared to sham-surgery. Females subjected to pTBI instead exhibited increased expression in all CA subregions 3 days post-injury, the only time point expression in CA2 was greater in females than in males. Immunohistochemical analysis confirmed neuronal CYP1B1 protein in all hippocampal subregions, while CPR was limited to CA1 and CA2. CYP1B1 mRNA is constitutively expressed in both sexes. In response to pTBI, females displayed a more urgent but brief regulatory response than males. This indicates there may be sex-dependent differences in CYP1B1 activity, possibly influencing inflammation and neuroprotection in pTBI.     

Molecular Evolution of a Steroid Hydroxylating Cytochrome P450 Using a Versatile Steroid Detection System for Screening

Molecular evolution is a powerful tool for improving or changing activities of enzymes for their use in biotechnological processes. Cytochromes P450 are highly interesting enzymes for biotechnological purposes because they are able to hydroxylate a broad variety of substrates with high regio- and stereoselectivity. One promising steroid hydroxylating cytochrome P450 for biotechnological applications is CYP106A2 from Bacillus megaterium ATCC 13368. It is one of a few known bacterial cytochromes P450 able to transform steroids such as progesterone and 11-deoxycortisol. CYP106A2 can be easily expressed in Escherichia coli with a high yield and can be reconstituted using the adrenal redox proteins, adrenodoxin and adrenodoxin reductase. We developed a simple screening assay for this system and performed random mutagenesis of CYP106A2, yielding variants with improved 11-deoxycortisol and progesterone hydroxylation activity. After two generations of directed evolution, we were able to improve the k (cat)/K (m) of the 11-deoxycortisol hydroxylation by a factor of more than four. At the same time progesterone conversion was improved about 1.4-fold. Mapping the mutations identified in catalytically improved CYP106A2 variants into the structure of a CYP106A2 model suggests that these mutations influence the mobility of the F/G loop, and the interaction with the redox partner adrenodoxin. The results show the evolution of a soluble steroid hydroxylase as a potential new catalyst for the production of steroidogenic compounds.