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。     

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。     

Purification of cytochrome P450 BM-3 as a monooxygenase

After investigating two anion-exchange resins, the purification factor and activity yields of P450 BM-3 were higher with Resource Q than with DEAE-Sepharose FF. Screening of HIC media showed that Source 15ISO was the most suitable for purification of P450 BM-3. An effective isolation and purification procedure of P450 BM-3 was developed and included three steps: 35%-70% saturation (NH(4))(2)SO(4) precipitation, Source 15ISO hydrophobic interaction chromatograph and Sephacryl S-200 gel filtration chromatography. Using this protocol, the purification factor and P450 BM-3 activity recovery was 13.5 and 13.7%, respectively.     

Hydrogen peroxide-mediated inactivation of microsomal cytochrome P450 during monooxygenase reactions

Cytochrome P450 can undergo inactivation following monooxygenase reactions in liver microsomes of untreated, phenobarbital and 3-methylcholanthrene-treated rats and rabbits. The acceleration of cytochrome P450 loss in the presence of catalase inhibitors (sodium azide, hydroxylamine) indicates that hydrogen peroxide is involved in hemoprotein degradation. It was revealed that cytochrome P450 is inactivated mainly by H₂O₂ formed through peroxy complex breakdown, whereas H₂O₂ formed via the dismutation of superoxide anions produces a slight inactivating effect. The hydrogen peroxide added outside or formed by a glucose-glucose oxidase system has less of an inactivating effect than H₂O₂ produced within the cytochrome P450 active center. Self-inactivation of cytochrome P450 during oxygenase reactions is highly specific. Other components of the monooxygenase system, such as cytochrome b₅, NADH- and NADPH-specific flavorproteins, undergo no inactivation. The alterations in phospholipid content and in the rate of lipid peroxidation were not observed as well. The inactivation of cytochrome P450 by H₂O₂ is the result of heme loss or destruction without cytochrome P420 formation. Such. a mechanism operates with different substrates and cytochrome P450 species catalyzing the partially coupled monooxygenase reactions.     

烟草细胞色素P450的基因组学分析

细胞色素P450是一类含血红素的单加氧酶超基因家族, 在植物多种代谢途径中起着重要作用。为了解烟草中的P450的种类和数量, 文章将植物代表性P450蛋白质序列与烟草基因组序列比对, 在烟草基因组中鉴定了44个P450家族共263个成员。将这些烟草P450基因与烟草表达序列标签(EST)比对, 发现173个成员有EST证据。通过与拟南芥中已知的P450蛋白序列比较, 分析了部分烟草P450蛋白序列的特征和二级结构。根据烟草基因芯片数据和部分基因的RT-PCR结果, 发现73个烟草P450基因能够在不同的生长发育时期表达, 其中部分基因具有组织特异性。这些研究结果为烟草P450基因功能的深入分析奠定了基础。     

Regio- and stereoselective regulation of monooxygenase activities by isoenzyme-selective phosphorylation of cytochrome P450

The phosphorylation of the two major phenobarbital-inducible cytochrome P450 isoenzymes IIB1 and IIB2 was increased in hepatocytes by the action of the membrane permeating cAMP derivatives N6-dibutyryl-cAMP and 8-thiomethyl-cAMP. Under these conditions the dealkylation of 7-pentoxyresorufin, a selective substrate of cytochrome P450IIB1 and P450IIB2 was markedly reduced. 16 beta-Hydroxylation of testosterone which is catalyzed specifically only by cytochrome P450IIB1 and IIB2 was strongly reduced; for 16 alpha-hydroxylation which is also catalyzed by cytochrome P450IIB1 and IIB2 but additionally by 3 further cytochrome P450 isoenzymes, this reduction was less pronounced; for the oxidation of the 17 beta-hydroxyl group which besides cytochromes P450IIB1 and IIB2 is additionally catalyzed not only by other cytochromes P450 but also by 17 beta-hydroxysteroid dehydrogenase there was a clear tendency of reduction which, however, no longer reached statistical significance. Hydroxylation at other positions of testosterone which are catalyzed by other cytochrome P450 isoenzymes were not significantly changed. Hence isoenzyme-selective phosphorylation of cytochrome P450 leads to a corresponding isoenzyme-selective modulation of monooxygenase activity which holds promise to be especially important as a fast regulation of the control of genotoxic metabolites.     

Transformation of fatty acids catalyzed by cytochrome P450 monooxygenase enzymes of Candida tropicalis

Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in beta-oxidation convert these substrates to long-chain alpha,omega-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is omega-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C(18:1)), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to omega-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C(14:0)) much more effectively. Both enzymes, in particular CYP52A17, also oxidized omega-hydroxy fatty acids, ultimately generating the alpha,omega-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for beta-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.     

Involvement of a cytochrome P450 monooxygenase in thaxtomin A biosynthesis by Streptomyces acidiscabies

The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3' of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as (1)H- and (13)C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide.     

Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis

The insecticidal loline alkaloids, produced by Neotyphodium uncinatum and related endophytes, are exo-1-aminopyrrolizidines with an ether bridge between C-2 and C-7. Loline alkaloids vary in methyl, acetyl, and formyl substituents on the 1-amine, which affect their biological activity. Enzymes for key loline biosynthesis steps are probably encoded by genes in the LOL cluster, which is duplicated in N. uncinatum, except for a large deletion in lolP2. The role of lolP1 was investigated by its replacement with a hygromycin B phosphotransferase gene. Compared to wild type N. uncinatum and an ectopic transformant, DeltalolP1 cultures had greatly elevated levels of N-methylloline (NML) and lacked N-formylloline (NFL). Complementation of DeltalolP1 with lolP1 under control of the Emericella nidulans trpC promoter restored NFL production. These results and the inferred sequence of LolP1 indicate that it is a cytochrome P450, catalyzing oxygenation of an N-methyl group in NML to the N-formyl group in NFL.     

The monooxygenase, peroxidase, and peroxygenase properties of cytochrome P450

This review examines the monooxygenase, peroxidase, and peroxygenase properties of cytochrome P450 (P450)1 enzymes and their mechanisms of action in archaeal, bacterial, and mammalian systems. In the P450 catalytic cycle, a transient iron oxo monooxygenating species is generated that reacts with substrate to produce a monooxygenated product. We describe results of early investigations that endeavored to trap and detect this elusive monooxygenating species, as well as results of experiments that attempted to generate and characterize this active oxidant spectroscopically after reacting ferric P450 enzymes with peroxy compounds (e.g. peroxides, peracids) or single oxygen atom donors (e.g. periodate, iodosobenzene). Surrogate oxidants were able to promote P450-catalyzed monooxygenations in a manner similar to that of O2/NAD(P)H, suggesting involvement of a common transitory monooxygenating species in the two pathways. This common P450 oxidant was characterized as a porphyrin radical iron(IV) oxo complex and assigned a Compound I structure (Por+FeIV=O) exhibiting a formal FeV oxidation state. Other reactive oxidants, such as the ferric oxenoid complex (PorFeIII=O), ferryloxy radical species (PorFeIV-O·), and perferryloxo entity (PorFeV=O), were also proposed to function as P450 monooxygenating species. We also discuss the possible involvement of the ferriperoxo (PorFeIII-OO-) and ferrihydroperoxo (PorFeIII-OOH) species as alternative oxidants in P450-mediated monooxygenation reactions.     

Engineering of the cytochrome P450 monooxygenase system for benzyl maltol hydroxylation

Maltol derivatives are utilized in a variety of fields due to their metal-chelating abilities, and modification of the 2-methyl side chain is known to effectively expand their functional diversity. In the present study, microbial enzymes were screened for hydroxylating activity towards the 2-methyl group in a maltol derivative, 3-benzyloxy-2-methyl-4-pyrone (BMAL). Novosphingobium sp. SB32149 was found to have the ability to convert BMAL into 3-benzyloxy-2-hydroxymethyl-4-pyrone (BMAL-OH). The enzymes responsible, a cytochrome P450 monooxygenase (P450nov), a ferredoxin (FDXnov), and a ferredoxin reductase (FDRnov), were identified in the SB32149 strain. In the reaction with recombinant Escherichia coli expressing P450nov, FDXnov, and FDRnov, BMAL-OH was successfully produced from BMAL. Moreover, using the directed evolution approach, four amino acid substitutions, L188P/F218L/L237M in P450nov and A10T in FDXnov, were found to enhance BMAL-OH production. Consequently, up to 5.2 g/L BMAL-OH was obtained from 8.0 g/L BMAL by bioconversion using a 250-mL jar fermenter, indicating that this strain may be useful for synthesis of maltol derivatives which could have potential applications in various fields.     

Binding of cytochrome P450 monooxygenase and lipoxygenase pathway products by heart fatty acid-binding protein

Arachidonic acid metabolism by lipoxygenases and cytochrome P450 monooxygenases produces regioisomeric hydroperoxyeicosatetraenoic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs), which serve as components of cell signaling cascades. Intracellular fatty acid-binding proteins (FABPs) may differentially bind these nonprostanoid oxygenated fatty acids, thus modulating their metabolism and activities. Vascular cells, which express heart FABP (H-FABP), utilize oxygenated fatty acids for regulation of vascular tone. Therefore, the relative affinities of H-FABP for several isomeric series of these compounds were measured by fluorescent displacement of 1-anilinonaphthalene-8-sulfonic acid (ANS). In general, H-FABP rank order affinities (arachidonic acid > EETs > HETEs > DHETs) paralleled reversed-phase high-performance liquid chromatography retention times, indicating that the differences in H-FABP affinity were determined largely by polarity. H-FABP displayed a similar rank order of affinity for compounds derived from linoleic acid. H-FABP affinity for 20-HETE [apparent dissociation constant (K(d)') of 0.44 microM] was much greater than expected from its polarity, indicating unique binding interactions for this HETE. H-FABP affinity for 5,6-EET and 11,12-EET (K(d)' of approximately 0.4 microM) was approximately 20-fold greater than for DHETs (K(d)' of approximately 8 microM). The homologous proteins, liver FABP and intestinal FABP, also displayed selective affinity for EET versus DHET. Thus, FABP binding of EETs may facilitate their intracellular retention whereas the lack of FABP affinity for DHETs may partially explain their release from cells. The affinity of H-FABP for EETs suggests that this family of intracellular proteins may modulate the metabolism, activities, and targeting of these potent eicosanoid biomediators.     

The cytochrome P450-containing monooxygenase of Trichosporon cutaneum: Occurrence and properties

Trichosporon cutaneum metabolizes glucose purely oxidatively and cytochrome P450 was not detected in the reduced CO-difference spectrum of whole cells. However, in the isolated microsomal fraction the corresponding monooxygenase was present as shown by the appearence of cytochrome P450, NADPH-cytochrome c (P450) reductase and cytochrome b₅. The absorption maximum of the terminal oxidase in the reduced CO-difference spectrum shifted between 447 and 448 nm. Derepression of biosynthesis of all components was achieved by transition of the cells from carbon- to oxygen-limited growth in continuous culture. The monooxygenase exhibited aminopyrine demethylation activity but not -hydroxylation activity of lauric acid. With respect to the growth limiting nutrient (carbon and oxygen respectively), mitochondrial cytochrome content showed an analogous behavior as cytochrome P450 and cytochrome b₅.     

Inhibition of cytochrome P-450-linked monooxygenase systems by naphthoquinones

Several naphthoquinones, except 2-hydroxy-1,4-naphthoquinone, were found to inhibit microsomal cytochrome P-450-linked monooxygenase activities in rabbit liver and human placenta. In particular, 5-hydroxy-1,4-naphthoquinone inhibited placental estrogen biosynthesis more effectively than it did hepatic drug oxidation reactions. There was little contribution by superoxide radicals to these enzyme inhibitions by naphthoquinones. Spectrophotometric studies revealed that naphthoquinones bind to the cytochrome P-450 component of the monooxygenase complex in both microsomal systems, suggesting that the inhibition is caused by direct interaction of these compounds with the heme.     

Engineering cytochrome P450 monooxygenase CYP 116B3 for high dealkylation activity

Cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber catalyzes the dealkylation of 7-ethoxycoumarin and the hydroxylation of substituted and unsubstituted aromatics. However, since activities were quite low, a combination of site-specific mutagenesis and directed evolution was applied to produce 7800 variants of CYP116B3, which were screened via a newly developed high-throughput screening system based on the dealkylation of 7-ethoxycoumarin catalyzed by recombinant E. coli. The best mutant was found after four rounds of directed evolution and had a 240-fold increased deethylation activity toward 7-ethoxycoumarin (223 nmol product/nmol P450.min) and a 10-fold increased demethylation activity toward 7-methoxycoumarin (9 nmol product/nmol P450.min).     

Influence of acetaminophen and trichloroethylene on liver cytochrome P450-dependent monooxygenase system

The aim of the study was to evaluate the effect of acetaminophen (APAP) and/or trichloroethylene (TRI) on the liver cytochrome P450-dependent monooxygenase system, CYP2E1 and CYP1A2 (two important P450 isoforms), and liver glutathione (GSH) content in rats. Rats were given three different doses of APAP (250, 500 and 1000 mg/kg b...) and then the above-mentioned parameters were measured for 48 h. The lowest APAP dose produced small changes in the cytochrome P450 content of liver. At 500 mg/kg APAP increased the cytochrome P450 content to 230% of the control. The inductive effect was seen at 1000 mg/kg dose but at 24 h and later. NADPH-cytochrome P450 reductase activity was the highest after the lowest dose of APAP, while after the highest dose it was equal to the control value. TRI increased both the cytochrome P450 content and the NADPH-cytochrome P450 reductase activity. When TRI was combined with APAP, both these parameters increased in the first hours of observation, but they returned to the control values at 24 h. When APAP was given at 250 mg/kg, GSH levels decreased to 55% of the control at 8 h and returned to the control values at 24 h. The higher doses of APAP decreased GSH levels more than the lowest dose, but after 24 h GSH levels did not differ from those of the control. When TRI was given at 250 mg/kg, the GSH levels decreased to 68% of the control at 2 h and then they increased gradually and tended to exceed the control values at 48 h. The effect of TRI combined with APAP on the level of GSH was virtually the same as that of APAP alone given at 500 mg/kg.     

Taxoid metabolism: Taxoid 14beta-hydroxylase is a cytochrome P450-dependent monooxygenase

The production of the anticancer drug Taxol in Taxus (yew) cell cultures is often accompanied by the formation of side-route polyoxygenated taxoid metabolites bearing a 14beta-hydroxyl group. The recent acquisition of several new semisynthetic taxoid intermediates enabled the screening of a family of Taxus cytochrome P450 cDNA clones for the 14beta-hydroxylase and additional taxoid oxygenases. The candidate cytochrome P450 clones were functionally expressed in yeast and tested by in vivo feeding of radiolabeled 5alpha-acetoxy-10beta-hydroxy taxadiene and 5alpha,13alpha-dihydroxy taxadiene. One clone efficiently and specifically transformed the 5alpha-acetoxy-10beta-ol, but not the 5alpha,13alpha-diol, to a more polar product with the chromatographic properties of a taxoid triol monoacetate, and the identity of this product was confirmed by spectroscopic means as 5alpha-acetoxy-10beta,14beta-dihydroxy taxadiene. Microsome preparation from the transformed yeast allowed characterization of this new hydroxylase, which was shown to resemble other cytochrome P450 taxoid hydroxylases with pH optimum at 7.5 and a K(m) value for the taxoid substrate of about 50 microM. Because Taxol is unsubstituted at C14, the 14beta-hydroxylase cannot reside on the pathway to the target drug but rather appears to be responsible for diversion of the pathway to 14-hydroxy taxoids that are prominent metabolites of Taxus cell cultures. Manipulation of this hydroxylase gene could permit redirection of the pathway to increase flux toward Taxol and could allow the preparation of 13alpha,14beta-hydroxy taxoids as new therapeutic agents.     

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.