定向进化细胞色素P450BM-3的研究

以来自于巨大芽孢杆菌的细胞色素P450BM-3为研究对象,采用随机突变和饱和定点突变定向进化技术对P450BM-3进行改造,通过突变体催化靛蓝显色的特性采用活性琼脂平板分析和96微孔板相结合的高通量筛选成功获得了几个具有更高催化性能的突变体。     

Features of cytochrome P450 evolution]

It is shown that the process of mutation in the CYP2 family of the superfamily of P450 cytochromes is species-specific (man, rat, and mouse). It is also shown that, within one species (rat), different families (CYP2 and CYP11) have different mutation spectra, indicating a high specificity of the mutation process for the families of cytochrome genes. A similar specificity was demonstrated for five families (CYP1, CYP2, CYP6, CYP7, CYP11) as compared with globins and prions. The analysis of the evolutionary mutation pattern, and the pattern of pseudogenes and damaged alleles of the CYP21 family (found in patients with congenital adrenal hyperplasia) does not confirm the widely accepted hypothesis that mutations arising in pseudogenes are transduced to normal alleles of the CYP21 gene through gene conversion.     

Improving the cytochrome P450 enzyme system for electrode-driven biocatalysis of styrene epoxidation

Cytochrome P450 enzymes catalyze a vast array of oxidative and reductive biotransformations that are potentially useful for industrial and pharmaceutical syntheses. Factors such as cofactor utilization and slow reaction rates for nonnatural substrates limit their large-scale usefulness. This paper reports several improvements that make the cytochrome P450cam enzyme system more practical for the epoxidation of styrene. NADH coupling was increased from 14 to 54 mol %, and product turnover rate was increased from 8 to 70 min(-1) by introducing the Y96F mutation to P450cam. Styrene and styrene oxide mass balance determinations showed different product profiles at low and high styrene conversion levels. For styrene conversion less than about 25 mol %, the stoichiometry between styrene consumption and styrene oxide formation was 1:1. At high styrene conversion, a second doubly oxidized product, alpha-hydroxyacetophenone, was formed. This was also the exclusive product when Y96F P450cam acted on racemic, commercially available styrene oxide. The alpha-hydroxyacetophenone product was suppressed in reactions where styrene was present at saturating concentrations. Finally, styrene epoxidation was carried out in an electroenzymatic reactor. In this scheme, the costly NADH cofactor and one of the three proteins (putidaredoxin reductase) are eliminated from the Y96F P450cam enzyme system.     

High diversity and complex evolution of fungal cytochrome P450 reductase: cytochrome P450 systems

Cytochrome P450 reductase (CPR) is the redox partner of P450 monooxygenases, involved in primary and secondary metabolism of eukaryotes. Two novel CPR genes, sharing 34% amino acid identity, were found in the filamentous ascomycete Cochliobolus lunatus. Fungal genomes were searched for putative CPR enzymes. Phylogenetic analysis suggests that multiple independent CPR duplication events occurred in fungi, whereas P450-CPR fusion occurred before the diversification of Dikarya and Zygomycota. Additionally, losses of methionine synthase reductase were found in certain fungal taxa; a truncated form of this enzyme was conserved in Pezizomycotina. In fungi, high numbers of cytochrome P450 enzymes, multiple CPRs, and P450-CPR fusion proteins were associated with filamentous growth. Evolution of multiple CPR-like oxidoreductases in filamentous fungi might have been driven by the complexity of biochemical functions necessitated by their growth form, as opposed to yeast.     

The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s

The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the ‘cytochrome P450 genesis locus’, where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution.     

High diversity and complex evolution of fungal cytochrome P450 reductase: cytochrome P450 systems.

Cytochrome P450 reductase (CPR) is the redox partner of P450 monooxygenases, involved in primary and secondary metabolism of eukaryotes. Two novel CPR genes, sharing 34% amino acid identity, were found in the filamentous ascomycete Cochliobolus lunatus. Fungal genomes were searched for putative CPR enzymes. Phylogenetic analysis suggests that multiple independent CPR duplication events occurred in fungi, whereas P450-CPR fusion occurred before the diversification of Dikarya and Zygomycota. Additionally, losses of methionine synthase reductase were found in certain fungal taxa; a truncated form of this enzyme was conserved in Pezizomycotina. In fungi, high numbers of cytochrome P450 enzymes, multiple CPRs, and P450-CPR fusion proteins were associated with filamentous growth. Evolution of multiple CPR-like oxidoreductases in filamentous fungi might have been driven by the complexity of biochemical functions necessitated by their growth form, as opposed to yeast.     

Reflections on sterol sidechain cleavage process catalyzed by cytochrome P450(scc)

The essay examines the evidence upon which the presently accepted version of the mechanism of the cytochrome P450(scc)-catalyzed-cleavage of the sidechain of cholesterol is based. This analysis indicates that the generally held view of the process (two consecutive hydroxylations, followed by cleavage of the resulting glycol) most likely does not describe the true mechanism. The available evidence can not be used to support this traditional notion. Two alternative hypotheses are proposed.     

First evidence that cytochrome P450 may catalyze both S-oxidation and epoxidation of thiophene derivatives

Oxidation of 2-phenylthiophene (2PT) by rat liver microsomes, in the presence of NADPH and glutathione (GSH), led to three kinds of metabolites whose structures were established by 1H NMR and mass spectrometry. The first ones were 2PT-S-oxide dimers formed by Diels-Alder type dimerization of 2PT-S-oxide, while the second ones were GSH adducts derived from the 1,4-Micha?l-type addition of GSH to 2PT-S-oxide. The third metabolites were GSH adducts resulting from a nucleophilic attack of GSH to the 4,5-epoxide of 2PT. Oxidation of 2PT by recombinant, human cytochrome P4501A1, in the presence of NADPH and GSH, also led to these three kinds of metabolites. These results provide the first evidence that cytochrome P450 may catalyze the oxidation of thiophene compounds with the simultaneous formation of two reactive intermediates, a thiophene-S-oxide and a thiophene epoxide.     

Colorimetric high-throughput assay for alkene epoxidation catalyzed by cytochrome P450 BM-3 variant 139-3

Cytochrome P450 BM-3 variant 139-3 is highly active in the hydroxylation of alkanes and fatty acids (AGlieder, ET Farinas, and FH Arnold, Nature Biotech 2002;20:1135-1139); it also epoxidizes various alkenes, including styrene. Here the authors describe a colorimetric, high-throughput assay suitable for optimizing this latter activity by directed evolution. The product of styrene oxidation by 139-3, styrene oxide, reacts with the nucleophile gamma-(4-nitrobenzyl)pyridine (NBP) to form a purple-colored precursor dye, which can be monitored spectrophotometrically in cell lysates. The sensitivity limit of this assay is 50-100 microM of product, and the detection limit for P450 BM-3 139-3 is ~0.2 microM of enzyme. To validate the assay, activities in a small library of random mutants were compared to those determined using an NADPH depletion assay for initial turnover rates.     

Control of the stereo-selectivity of styrene epoxidation by cytochrome P450 BM3 using structure-based mutagenesis

The potential of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium for biocatalysis and biotechnological application is widely acknowledged. The catalytic and structural analysis of the Ala82Phe mutant of P450 BM3 has shown that filling a hydrophobic pocket near the active site improved the binding of small molecules, such as indole (see Huang et al., J. Mol. Biol., 2007, 373, 633) and styrene. In this paper, additional mutations at Thr438 are shown to decrease the binding of and catalytic activity towards laurate, whereas they significantly increased the stereo-specificity of styrene epoxidation. Production of R-styrene oxide with 48% and 64% e.e., respectively, was achieved by the Ala82Phe-Thr438Leu and Ala82Phe-Thr438Phe mutants. These structure-based mutants of P450 BM3 illustrate the promise of rational design of synthetically useful biocatalysts for regio- and stereo- specific mono-oxygenation reactions.     

Directed molecular evolution of cytochrome c peroxidase

Cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae was subjected to directed molecular evolution to generate mutants with increased activity against the classical peroxidase substrate guaiacol, thus changing the substrate specificity of CCP from the protein cytochrome c to a small organic molecule. After three rounds of DNA shuffling and screening, mutants were isolated which possessed a 300-fold increased activity against guaiacol and an up to 1000-fold increased specificity for this substrate relative to that for the natural substrate. In all of the selected mutants, the distal arginine (Arg48), which is fully conserved in the superfamily of peroxidases, was mutated to histidine, showing that this mutation plays a key role in the significant increase in activity against phenolic substrates. The results suggest that, in addition to stabilizing the reactive intermediate compound I, the distal arginine plays an important role as a gatekeeper in the active site of CCP, controlling the access to the ferryl oxygen and the distal histidine. Other isolated mutations increase the general reactivity of the peroxidase or increase the intracellular concentration of the active holo form, allowing their selection under the employed screening conditions. The results illustrate the ability of directed molecular evolution technologies to deliver solutions to biochemical problems that would not be readily predicted by rational design.     

Thermophilic cytochrome P450 enzymes

Thermophilic cytochrome P450 enzymes are of potential interest from structural, mechanistic, and biotechnological points of view. The structures and properties of two such enzymes, CYP119 and CYP175A1, have been investigated and provide the foundation for future work on thermophilic P450 enzymes.     

Rapid birth-death evolution specific to xenobiotic cytochrome P450 genes in vertebrates

Genes vary greatly in their long-term phylogenetic stability and there exists no general explanation for these differences. The cytochrome P450 (CYP450) gene superfamily is well suited to investigating this problem because it is large and well studied, and it includes both stable and unstable genes. CYP450 genes encode oxidase enzymes that function in metabolism of endogenous small molecules and in detoxification of xenobiotic compounds. Both types of enzymes have been intensively studied. My analysis of ten nearly complete vertebrate genomes indicates that each genome contains 50-80 CYP450 genes, which are about evenly divided between phylogenetically stable and unstable genes. The stable genes are characterized by few or no gene duplications or losses in species ranging from bony fish to mammals, whereas unstable genes are characterized by frequent gene duplications and losses (birth-death evolution) even among closely related species. All of the CYP450 genes that encode enzymes with known endogenous substrates are phylogenetically stable. In contrast, most of the unstable genes encode enzymes that function as xenobiotic detoxifiers. Nearly all unstable CYP450 genes in the mouse and human genomes reside in a few dense gene clusters, forming unstable gene islands that arose by recurrent local gene duplication. Evidence for positive selection in amino acid sequence is restricted to these unstable CYP450 genes, and sites of selection are associated with substrate-binding regions in the protein structure. These results can be explained by a general model in which phylogenetically stable genes have core functions in development and physiology, whereas unstable genes have accessory functions associated with unstable environmental interactions such as toxin and pathogen exposure. Unstable gene islands in vertebrates share some functional properties with bacterial genomic islands, though they arise by local gene duplication rather than horizontal gene transfer.     

Moonlighting cytochrome P450 monooxygenases

Recently, cytochrome P450 170A1 (CYP170A1) has been found to be a bifunctional protein, which catalyzes both monooxygenase activity and terpene synthase activity by two distinct active sites in the well-established P450 protein structure. Therefore, CYP170A1 is identified clearly as a moonlighting protein. The known activities of a small number of the 13,000 members of the P450 superfamily fall into two general classes: promiscuous enzymes that are not considered as moonlighting and forms that participate in biosynthesis of endogenous compounds, such as steroids, vitamins and play different roles in different tissues, sometimes being moonlighting enzymes. Here, we review examples of moonlighting P450, which add to our understanding of the large CYP superfamily.     

Spectroelectrochemistry of cytochrome P450cam

The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described. The electrodes were modified with either 4,4(')-dithiodipyridin or sodium dithionite. Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV-visible absorption spectra were recorded. Reversible oxidation and reduction could be observed with both 4,4(')-dithiodipyridin and dithionite modified electrodes. A formal potential (E(0')) of -373mV vs Ag/AgCl 1M KCl was determined. The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis.     

Forty years of cytochrome P450

The term "cytochrome P450" first appeared in literature in 1962. It was a microsomal membrane-bound hemoprotein without known physiological functions at that time and was characterized by a unique 450-nm optical absorption peak of its carbon monoxide-bound form, which was originally reported as the spectrum of a novel "microsomal carbon monoxide-binding pigment" in 1958. Elucidation of its function as the oxygenase in 1963 triggered a rapid expansion of research on this hemoprotein. Annual numbers of the published papers dealing with cytochrome P450, which were listed in Biological Abstracts, increased from 60 in 1970 to 500 in 1980, 900 in 1990, and 1500 in 1997. Cytochrome P450 is now regarded as the collective name of a large family of hemoproteins, "cytochrome P450 superfamily, "which seems to have diversified from a single ancestral protein to many forms during the course of biological evolution and is distributed widely among various forms of life from animals and plants to fungi and bacteria. Multicellular eukaryotic organisms including animals and plants have about 100 or more P450 genes in their genomes, and those many P450 genes are expressed tissue specifically and developmental stage specifically, indicating their diverse physiological functions. In mammals, various P450s participate in the biosynthesis and metabolism of sterols and steroid hormones and the metabolism of various lipid biofactors including eicosanoids, vitamin D3, and retinoids. Oxidative metabolism of foreign hydrophobic compounds as the first step of their excretion from the animal body is apparently another major function of cytochrome P450, which protects animals from noxious foreign compounds, man-created and natural.     

Improved cloning and expression of cytochrome P450s and cytochrome P450 reductase in yeast

Combination of the pYeDP60 yeast expression system with a modified version of the improved uracil-excision (USER) cloning technique provides a new powerful tool for high-throughput expression of eukaryotic cytochrome P450s. The vector presented is designed to obtain an optimal 5' untranslated sequence region for yeast (Kozak consensus sequence), and has been tested to produce active P450s and NADPH-cytochrome P450 oxidoreductase (CPR) after 5' end silent codon optimization of the cDNA sequences. Expression of two plant cytochrome P450s, Sorghum bicolor CYP79A1 and CYP71E1, and S. bicolor CPR2 using the modified pYeDP60 vector in all three cases produced high amounts of active protein. High-throughput functional expression of cytochrome P450s have long been a troublesome task due to the workload involved in cloning of each individual P450 into a suitable expression vector. The redesigned yeast P450 expression vector (pYeDP60u) offers major improvements in cloning efficiency, speed, fidelity, and simplicity. The modified version of the USER cloning system provides great potential for further development of other yeast vectors, transforming these into powerful high-throughput expression vectors.     

Computer-based substrate specificity prediction for cytochrome P450

Cytochrome P450 is an important class of enzymes metabolizing numerous drugs. The composition and activity of these enzymes determine distribution of drug in the body, their pharmacological and toxic effects. Thus, prediction of the fate of compounds in the body is required at early stages of the development of new drugs. Different isoforms of cytochrome P450 can oxidize a wide range of chemical compounds and their substrate specificity does not correlate with their taxonomical classification. In this review we consider the main methods of cytochrome P450 substrate specificity prediction. These methods are subdivided by primary information used in the analysis: amino acid sequence based prediction, ligand-based (pharmacophore and QSAR models, expert systems) and structure-based (molecular docking, affinity prediction, interaction energy estimation) methods. The common problems complicating cytochrome P450 substrate prediction and advantages and disadvantages of these methods are discussed.