Identification of a new class of cytochrome P450 from a Rhodococcus sp

A degenerate set of PCR primers were used to clone a gene encoding a cytochrome P450 (the P450RhF gene) from Rhodococcus sp. strain NCIMB 9784 which is of unique primary structural organization. Surprisingly, analysis of the translation product revealed that the P450 is fused to a reductase domain at the C terminus which displays sequence conservation for dioxygenase reductase proteins. The reductase partner comprises flavin mononucleotide- and NADH-binding motifs and a [2Fe2S] ferredoxin-like center. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a soluble form. A recombinant strain of E. coli was able to mediate the O dealkylation of 7-ethoxycoumarin in good yield, despite the absence of any recombinant redox proteins. This unprecedented finding leads us to propose that P450RhF represents the first example of a new class of cytochromes P450 in which the reducing equivalents are supplied by a novel reductase in a fused arrangement.     

Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319

A new member of class IV of cytochrome P450 monooxygenases was identified in Rhodococcus ruber strain DSM 44319. As the genome of R. ruber has not been sequenced, a P450-like gene fragment was amplified using degenerated primers. The flanking regions of the P450-like DNA fragment were identified by directional genome walking using polymerase chain reaction. The primary protein structure suggests a natural self-sufficient fusion protein consisting of ferredoxin, flavin-containing reductase, and P450 monooxygenase. The only flavin found within the enzyme was riboflavin 5′-monophosphate. The enzyme was successfully expressed in Escherichia coli, purified and characterized. In the presence of NADPH, the P450 monooxygenase showed hydroxylation activity towards polycyclic aromatic hydrocarbons naphthalene, indene, acenaphthene, toluene, fluorene, m-xylene, and ethyl benzene. The conversion of naphthalene, acenaphthene, and fluorene resulted in respective ring monohydroxylated metabolites. Alkyl aromatics like toluene, m-xylene, and ethyl benzene were hydroxylated exclusively at the side chains. The new enzyme’s ability to oxidize such compounds makes it a potential candidate for biodegradation of pollutants and an attractive biocatalyst for synthesis.     

Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784

Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites for a P450, a linker sequence, and the P450RhF reductase domain, in this order. The known P450 genes, encoding P450cam from Pseudomonas putida (CYP101A) and P450bzo from an environmental metagenome library (CYP203A), were expressed on vector pRED as soluble fusion enzymes with their natural spectral features in Escherichia coli. These E. coli cells expressing the P450cam and P450bzo genes could convert (+)-camphor and 4-hydroxybenzoate into 5-exo-hydroxycamphor and protocatechuate (3,4-dihydroxybenzoate), respectively (the expected products). Using this system, we also succeeded in directly identifying the function of P450 CYP153A as alkane 1-monooxygenase for the first time, i.e., E. coli cells expressing a P450 CYP153A gene named P450balk, which was isolated form Alcanivorax borkumensis SK2, converted octane into 1-octanol with high efficiency (800 mg/l). The system presented here may be applicable to the functional identification of a wide variety of bacterial cytochromes P450.     

Improved expression of recombinant cytochrome P450 monooxygenase in Escherichia coli for asymmetric oxidation of sulfides

Escherichia coli BL21 as production strain for the production of cytochrome P450 monooxygenase (P450SMO) from Rhodococcus sp. in high yields was developed. The expression was first optimized with a series of flask experiments testing several key parameters for their influence on the expression level and enzyme activity. The optimal process parameters found in the flask experiments were verified in a cultivation process in a 5-L bioreactor. Glycerol proved to be superior over glucose as carbon source. Low dissolved oxygen (DO) concentration (<10%) during expression was found to be critical for active P450s production, resulting in expression level of 400 nM for P450SMO. Intact cells were used to establish an efficient bioconversion system for the production of sulfoxidation product. With p-chlorothioanisole as a representative substrate, the desired product (S-sulfoxide) was afforded with 99% ee and highest production of 130 mg/L within 12 h.     

Spectroscopic characterization of a newly isolated cytochrome P450 from Rhodococcus rhodochrous.

Cytochrome P450 (P450) from Rhodococcus rhodochrous have been characterized through circular dichroism and nuclear magnetic resonance (NMR) spectroscopy, both in the substrate-free and substrate-bound forms. The data are compared with those of P450cam and indicate a close similarity of the structure of the active site in the two proteins. The substrate-free species contains low-spin iron(III), while the 2-ethoxyphenol bound species contains high-spin iron(III). The substrate is in slow exchange on the NMR time scale. The binding of CN- has been investigated and the final adduct characterized through NMR spectra. Nuclear relaxation times of the isotropically shifted signals turn out to be shorter than in other heme proteins, both in the high- and in the low-spin species. This is the result of longer electron relaxation times in P450s than in peroxidases and metmyoglobin. This property, as well as the electron paramagnetic resonance (EPR) spectrum of the substrate-free form, are discussed in terms of the presence of the cysteine as the fifth ligand of the iron ion instead of a histidine as it occurs in peroxidases and myoglobin.     

Engineering of heterologous cytochrome P450 in Acinetobacter sp.: application for pollutant degradation

Many organisms do not contain the necessary biochemical armoury to carry out the initial oxidative attack of many pollutant chemicals. In the present study, Acinetobacter sp. strain BD413 has been genetically engineered to express the cytochrome P450 xenobiotic-metabolising enzyme CYP105D1 from Streptomyces griseus that has in its repertoire a diverse array of organic pollutants. Further, it is shown that the transformed Acinetobacter calcoaceticus strain BD413 can grow on pollutants unlike control bacteria not expressing cytochrome P450 and that was reflected in release of radiolabel with growth on radiolabelled chlortoluron. We show that cytochrome P450 can enhance the biodegrading repertoire of A. calcoaceticus and discuss the application of such results to bioremediation strategies.     

Biocatalytic conversion of avermectin to 4"-oxo-avermectin: heterologous expression of the ema1 cytochrome P450 monooxygenase

The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1.     

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。     

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.     

Identification and heterologous expression of the cytochrome P450 oxidoreductase from the white-rot basidiomycete Coriolus versicolor

A cDNA encoding cytochrome P450 oxidoreductase (CPR) from the lignin-degrading basidiomycete Coriolus versicolor was identified using RT-PCR. The full-length cDNA consisted of 2,484 nucleotides with a poly(A) tail, and contained an open reading frame. The G+C content of the cDNA isolated was 60%. A deduced protein contained 730 amino acid residues with a calculated molecular weight of 80.7 kDa. The conserved amino acid residues involved in functional domains such as FAD-, FMN-, and NADPH-binding domains, were all found in the deduced protein. A phylogenetic analysis demonstrated that C. versicolor CPR is significantly similar to CPR of the basidiomycete Phanerochaete chrysosporium and that they share the same major branch in the fungal cluster. A recombinant CPR protein was expressed using a pET/ Escherichia coli system. The recombinant CPR protein migrated at 81 kDa on SDS polyacrylamide gel electrophoresis. It exhibited an NADPH-dependent cytochrome c reducing activity.     

昆虫细胞色素P450基因的多样性、进化及表达调控

细胞色素P450单加氧酶（cytochrome P450 monooxygenases, P450s）是由多个功能相关的亚铁血红素 硫醇盐蛋白基因组成的一个基因超家族, 在各种内源和外源物质的代谢中起着主要作用。目前GenBank中注册的昆虫P450基因序列已超过1 000个, 其中双翅目占序列总数的74%, 鳞翅目占序列总数的16%。而昆虫P450基因序列已克隆的全长序列中大部分属于CYP4和CYP6家族, 两个家族成员分别占总数的20%和45%。利用GenBank中现已注册的昆虫P450基因的cDNA全长序列进行比对并绘制进化树, 揭示不同种类昆虫P450的亲缘关系。结果显示基于P450基因的昆虫部分目的进化关系与大部分先前依据其他分子数据或形态分类学得到的昆虫系统进化关系基本吻合。现有研究表明, 细胞色素P450基因的表达可能受顺式作用元件(cis-acting element)、反式作用因子(trans-acting factor)或两者共同调控, 调控可能涉及转录增强的转录机制或mRNA稳定性增加的转录后机制。     

Increasing expression of P450 and P450-reductase proteins from monocots in heterologous systems

Monocotyledonous crop plants are usually more resistant to herbicides than grass weeds and most dicots. Their resistance to herbicides is mediated in many cases by P450 oxygenases. Monocots thus constitute an appealing source of P450 enzymes for manipulating herbicide resistance and recombinant forms of the major xenobiotic metabolizing mooxygenases are potential tools for the optimization of new active molecules. We report here the isolation and functional characterization of the first P450 and P450 reductase coding sequences from wheat. The first attempts at expressing these cDNAs in yeast and tobacco led to levels of protein, which were extremely low, often not even detectable. The wheat P450 cDNAs were efficiently transcribed, but no protein or activity was found. Wheat coding sequences, like those of other monocots, are characterized by a high GC content and by a related strong bias of codon usage, different from that observed in yeast or dicots. Complete recoding of genes being costly, the reengineering their 5'-end using a single PCR megaprimer designed to comply with codon usage of the host was attempted. It was sufficient to relieve translation inhibition and to obtain good levels of protein expression. The same strategy also resulted in a dramatic increase in protein expression in tobacco. A basis for the success of such a partial recoding strategy, much easier and cheaper than complete recoding of the cDNA, is proposed.     

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.     

Hepatic cytochrome P450 reductase-null mice reveal a second microsomal reductase for squalene monooxygenase

Squalene monooxygenase is a microsomal enzyme that catalyzes the conversion of squalene to 2,3(s)-oxidosqualene, the immediate precursor to lanosterol in the cholesterol biosynthesis pathway. Unlike other flavoprotein monooxygenases that obtain electrons directly from NAD(P)H, squalene monooxygenase requires a redox partner, and for many years it has been assumed that NADPH-cytochrome P450 reductase is this requisite redox partner. However, our studies with hepatic cytochrome P450-reductase-null mice have revealed a second microsomal reductase for squalene monooxygenase. Inhibition studies with antibody to P450 reductase indicate that this second reductase supports up to 40% of the monooxygenase activity that is obtained with microsomes from normal mice. Studies carried out with hepatocytes from CPR-null mice demonstrate that this second reductase is active in whole cells and leads to the accumulation of 24-dihydrolanosterol; this lanosterol metabolite also accumulates in the livers of CPR-null mice, indicating that cholesterol synthesis is blocked at lanosterol demethylase, a cytochrome P450.     

High-level heterologous expression of fungal cytochrome P450s in Escherichia coli

A thorough understanding of the sequence–structure–function relationships of cytochrome P450 (P450) is necessary to better understand the metabolic diversity of living organisms. Significant amounts of pure enzymes are sometimes required for biochemical studies, and their acquisition often relies on the possibility of their heterologous expression. In this study, we performed extensive heterologous expression of fungal P450s in Escherichia coli using 304 P450 isoforms. Using large-scale screening, we confirmed that at least 27 P450s could be expressed with/without simple sequence deletion at the 5′ end of cDNAs, which encode the N-terminal hydrophobic domain of the enzyme. Moreover, we identified N-terminal amino acid sequences that can potentially be used to construct chimeric P450s, which could dramatically improve their expression levels even when the expression of the wild-type sequence was unpromising. These findings will help increase the chance of heterologous expression of a variety of fungal and other eukaryotic membrane-bound P450s in E. coli.     

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.     

Cytochrome P450 monooxygenase from Clostridium acetobutylicum: a new alpha-fatty acid hydroxylase

Cytochrome P450 monooxygenase from the anaerobic microorganism Clostridium acetobutylicum (CYP152A2) has been produced in Escherichia coli. CYP152A2 was shown to bind a broad range of saturated and unsaturated fatty acids and corresponding methyl esters and demonstrated a high peroxygenase activity of up to 200min(-1) with myristic acid. Although a high concentration of hydrogen peroxide of 200microM was necessary for high activities of the enzyme, it led to a fast enzyme inactivation within 2-4min. This might reflect the natural function of CYP152A2 as a rapid hydrogen peroxide scavenging enzyme. In two different reconstituted systems with NADPH, CYP152A2 was able to convert 10 times more substrate, if provided with flavodoxin and flavodoxin reductase from E. coli and even 30-40 times more substrate with the CYP102A1-reductase from Bacillus megaterium. According to the clear preference for hydroxylation at alpha-position, CYP152A2 can be referred to as fatty acid alpha-hydroxylase.