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1.
P450 BM-3是一种具有工业化应用潜力的单加氧酶,可催化饱和脂肪酸羟基化。为提高其在大肠杆菌宿主中的可溶性表达水平,采用乳糖作为诱导剂对P450 BM-3的诱导表达条件进行研究。结果发现:在大肠杆菌的OD600达到0.7~1.5时,添加2.0 g/L的乳糖、30℃诱导10 h可获得最佳诱导效果。与IP TG的诱导效果对比发现:采用乳糖作诱导剂时,菌体生物量提高1.09倍,目标蛋白量提升2.13倍,蛋白包涵体的比例则降低至10%。研究结果表明:乳糖可显著提升P450 BM-3在大肠杆菌中的重组表达水平,并且能够促进p450 BM-3的可溶性表达。 相似文献
2.
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。 相似文献
3.
催化吲哚生成靛蓝的细胞色素P450BM-3 定向进化研究 总被引:6,自引:0,他引:6
以催化吲哚产生的靛蓝在 630 nm 处具有特殊的吸收峰为高通量筛选指标,将来源于 Bacillus megaterium 的细胞色素 P450BM-3 单加氧酶的基因序列用易错聚合酶链式反应进行定向进化,通过多轮突变,在原有的能产靛蓝的高活力突变酶的基础上成功获得了三个高于亲本酶的突变酶,突变酶的酶活分别是亲本酶的 6.6 倍 (hml001) , 9.6 倍 (hml002) 和 5.3 倍 (hml003) ,并对突变酶的动力学参数进行了分析 . 突变酶 DNA 测序的结果表明, hml001 含有一个有义氨基酸置换 I39V , hml002 含有三个有义氨基酸置换 D168N , A225V , K440N , hml003 含有一个有义氨基酸置换 E435D ,这些突变位点有些远离底物结合部位,有些位于底物结合部位 . 相似文献
4.
Quorum sensing is the process by which bacteria alter gene regulation in response to their population density. The enzymatic inactivation of quorum signals has shown promise for use in genetically modified organisms resistant to pathogens. We recently characterized the ability of a cytochrome P450, P450BM-3, to oxidize the quorum sensing signals known as acyl homoserine lactones. The oxidation of the acyl homoserine lactones reduced their activity as quorum signals. The enzyme also oxidized the inactive lactonolysis products, acyl homoserines. The enzyme showed similar binding affinity for the acyl homoserine lactones and acyl homoserines. The latter reaction may lead to problems when lactonases and the P450-dependent system are used in tandem, as oxidation of the acyl homoserines produced by lactonolysis in vivo may compete with acyl homoserine lactone oxidation by the cytochrome P450. We report here that a single mutation (R47S) in P450BM-3 is capable of increasing the acyl homoserine lactone: acyl homoserine substrate binding selectivity of the enzyme nearly 250-fold, reducing the potential for competition by acyl homoserines and significantly enhancing the potential for use of P450BM-3 as part of a pathogen resistance system in genetically modified crops. 相似文献
5.
Sulistyaningdyah WT Ogawa J Li QS Shinkyo R Sakaki T Inouye K Schmid RD Shimizu S 《Biotechnology letters》2004,26(24):1857-1860
The metabolism of polychlorinated dibenzo-p-dioxins by cytochrome P450 BM-3 from Bacillus megaterium and a mutant enzyme of it (AL4V; Ala74Gly, Phe87Val, Leu188Gln triple mutant) was examined. Both purified enzymes metabolized 1-monochloro-, 2,3-dichloro-, and 2,3,7-trichloro-dibenzo-p-dioxin, but not 2,3,7,8-tetrachloro-dibenzo-p-dioxin. The mutant AL 4V had 2–12 times higher activity than the wild-type P450 BM-3 towards polychlorinated dibenzo-p-dioxins. The products were hydroxylated at an unsubstituted position and/or showing migration of the chloride and were less toxic derivatives with lower than 10% toxicity of the original compounds.Revisions requested 26 August 2004; Revisions received 15 October 2004 相似文献
6.
Linda Owers Narhi Long-Ping Wen Armand J. Fulco 《Molecular and cellular biochemistry》1988,79(1):63-71
Summary In two previous reports (Narhi LO, Fulco AJ, J. Biol. Chem. 261: 7160–7169, 1986; Ibid., 262: 6683–6690, 1987) we described the characterization of a catalytically self-sufficient 119000-dalton P-450 cytochrome that was induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O2, this polypeptide (cytochrome P-450BM-3) catalyzed the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase was cloned into Escherichia coli and the clone harboring the recombinant plasmid produced a protein that behaved electrophoretically and immunochemically like the B. megaterium enzyme (Wen LP, Fulco AJ, J. Biol. Chem. 262: 6676–6682, 1987). We have now compared authentic P-450BM-3 from B. megaterium and putative P-450BM-3 isolated from transformed E. coli and have found them to be indistinguishable with respect to chromatographic and electrophoretic behavior, reaction with specific antibody, prosthetic group (heme, FAD and FMN) analyses, spectra, enzymology, limited trypsin proteolysis and partial amino acid sequencing. We thus conclude that the P-450 cytochrome expressed by the transformed E. coli is essentially identical to native P-450BM-3 induced by barbiturates in B. megaterium. The evidence furthermore suggests that the primary amino acid sequence of this complex protein is alone sufficient to direct the proper integration of the three prosthetic groups and to specify folding of the polypeptide into the correct tertiary structure.Abbreviations SDS
Sodium Dodecylsulfate
- PAGE
Polyacrylamide Gel Electrophoresis
- HPLC
High Performance Liquid Chromatography 相似文献
7.
We used a rapid scanning stop-flow technique to study the kinetics of reduction of cytochrome P450 3A4 (CYP3A4) by the flavin domain of cytochrome P450-BM3 (BMR), which was shown to form a stoichiometric complex (KD = 0.48 μM) with CYP3A4. In the absence of substrates only about 50% of CYP3A4 was able to accept electrons from BMR. Whereas the high-spin fraction was completely reducible, the reducibility of the low-spin fraction did not exceed 42%. Among four substrates tested (testosterone, 1-pyrenebutanol, bromocriptine, or α-naphthoflavone (ANF)) only ANF is capable of increasing the reducibility of the low-spin fraction to 75%. Our results demonstrate that the pool of CYP3A4 is heterogeneous, and not all P450 is competent for electron transfer in the complex with reductase. The increase in the reducibility of the enzyme in the presence of ANF may represent an important element of the mechanism of action of this activator. 相似文献
8.
We studied the kinetics of NADPH-dependent reduction of human CYP3A4 incorporated into Nanodiscs (CYP3A4-ND) and proteoliposomes in order to probe the effect of P450 oligomerization on its reduction. The flavin domain of cytochrome P450-BM3 (BMR) was used as a model electron donor partner. Unlike CYP3A4 oligomers, where only 50% of the enzyme was shown to be reducible by BMR, CYP3A4-ND could be reduced almost completely. High reducibility was also observed in proteoliposomes with a high lipid-to-protein ratio (L/P = 910), where the oligomerization equilibrium is displaced towards monomers. In contrast, the reducibililty in proteoliposomes with L/P = 76 did not exceed 55 ± 6%. The effect of the surface density of CYP3A4 in proteoliposomes on the oligomerization equilibrium was confirmed with a FRET-based assay employing a cysteine-depleted mutant labeled on Cys-468 with BODIPY iodoacetamide. These results confirm a pivotal role of CYP3A4 oligomerization in its functional heterogeneity. Furthermore, the investigation of the initial phase of the kinetics of CYP3A4 reduction showed that the addition of NADPH causes a rapid low-to-high-spin transition in the CYP3A4-BMR complex, which is followed by a partial slower reversal. This observation reveals a mechanism whereby the CYP3A4 spin equilibrium is modulated by the redox state of the bound flavoprotein. 相似文献
9.
Brian Gemzik Denise Greenway Cheryl Nevins Andrew Parkinson 《Journal of biochemical and molecular toxicology》1992,7(1):43-52
We recently reported that antibody against purified P450 3A1 (P450p) recognizes two electrophoretically distinct proteins (50 and 51 kDa) in liver microsomes from male and female rats, as determined by Western immunoblotting. Depending on the source of the liver microsomes, the 51-kDa protein corresponded to 3A1 and/or 3A2 which could not be resolved by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. The other protein (50 kDa) appears to be another member of the P450 IIIA gene family. Both proteins were markedly intensified in liver microsomes from male or female rats treated with pregnenolone-16α-carbonitrile, dexamethasone, troleandomycin, or chlordane. In contrast, treatment of male or female rats with phenobarbital intensified only the 51-kDa protein. Treatment of male rats with Aroclor 1254 induced the 51-kDa protein, but suppressed the 50-kDa form. In addition to their changes in response to inducers, the 50- and 51-kDa proteins also differed in their developmental expression. For example, the 50-kDa protein was not expressed until weaning (3 weeks), whereas the 51-kDa protein was expressed even in 1-week-old rats. At puberty (between weeks 5 and 6), the levels of the 50-kDa and 51-kDa proteins markedly declined in female but not in male rats, which introduced a large sex difference (male > female) in the levels of both proteins. Changes in the level of the 51-kDa protein were paralleled by changes in the rate of testosterone 2β, 6β-, and 15β-hydroxylation. In male rats, the marked increase in the levels of the 50-kDa protein between weeks 2 and 3 coincided with a three- to four fold increase in the rate of testosterone 2β-, 6β-, and 15β-hydroxylation, which suggests that the 50-kDa protein catalyzes the same pathways of testosterone oxidation as the 51-kDa protein. However, this developmental increase in testosterone oxidation may have resulted from an activation of the 51-kDa 3A protein. These results indicate that the two electrophoretically distinct proteins recognized by antibody against P450 3A1 are regulated in a similar but not identical manner, and suggest that the 51-kDa 3A protein is the major microsomal enzyme responsible for catalyzing the 2β-, 6β-, and 15β-hydroxylation of testosterone. 相似文献
10.
McArthur AG Hegelund T Cox RL Stegeman JJ Liljenberg M Olsson U Sundberg P Celander MC 《Journal of molecular evolution》2003,57(2):200-211
Cytochrome P450 genes (CYP) constitute a superfamily with members known from the Bacteria, Archaea, and Eukarya. The CYP3 gene family includes the CYP3A and CYP3B subfamilies. Members of the CYP3A subfamily represent the dominant CYP forms expressed in the digestive and respiratory tracts of vertebrates. The CYP3A enzymes metabolize a wide variety of chemically diverse lipophilic organic compounds. To understand vertebrate CYP3 diversity better, we determined the killifish (Fundulus heteroclitus) CYP3A30 and CYP3A56 and the ball python (Python regius) CYP3A42 sequences. We performed phylogenetic analyses of 45 vertebrate CYP3 amino acid sequences using a Bayesian approach. Our analyses indicate that teleost, diapsid, and mammalian CYP3A genes have undergone independent diversification and that the ancestral vertebrate genome contained a single CYP3A gene. Most CYP3A diversity is the product of recent gene duplication events. There is strong support for placement of the guinea pig CYP3A genes within the rodent CYP3A diversification. The rat, mouse, and hamster CYP3A genes are mixed among several rodent CYP3A subclades, indicative of a complex history involving speciation and gene duplication. Phylogenetic analyses suggest two CYP3A gene duplication events early in rodent history, with the rat CYP3A9 and mouse Cyp3a13 clade having a sister relationship to all other rodent CYP3A genes. In primate history, the human CYP3A43 gene appears to have a sister relationship to all other known primate CYP3A genes. Other, more recent gene duplications are hypothesized to have occurred independently within the human, pig, rat, mouse, guinea pig, and fish genomes. Functional analyses suggest that gene duplication is strongly tied to acquisition of new function and that convergent evolution of CYP3A function may be frequent among independent gene copies.
Current address (Rachel L. Cox): Laboratory of Aquatic Biomedicine, Marine Biology Laboratory, Woods Hole, MA 02543, USA 相似文献
11.
Charles A. Flentge John T. Randolph Peggy P. Huang Larry L. Klein Kennan C. Marsh John E. Harlan Dale J. Kempf 《Bioorganic & medicinal chemistry letters》2009,19(18):5444-5448
The HIV protease inhibitor ritonavir (RTV) is also a potent inhibitor of the metabolizing enzyme cytochrome P450 3A (CYP3A) and is clinically useful in HIV therapy in its ability to enhance human plasma levels of other HIV protease inhibitors (PIs). A novel series of CYP3A inhibitors was designed around the structural elements of RTV believed to be important to CYP3A inhibition, with general design features being the attachment of groups that mimic the P2–P3 segment of RTV to a soluble core. Several analogs were found to strongly enhance plasma levels of lopinavir (LPV), including 8, which compares favorably with RTV in the same model. Interestingly, an inverse correlation between in vitro inhibition of CYP3A and elevation of LPV was observed. The compounds described in this study may be useful for enhancing the pharmacokinetics of drugs that are metabolized by CYP3A. 相似文献
12.
Cytochrome P450 BM-3 from Bacillus megaterium is an extensively studied enzyme for industrial applications. A major focus of current protein engineering research is directed to improving the catalytic performance of P450 BM-3 toward nonnatural substrates of industrial importance in the presence of organic solvents or cosolvents. For the latter reason, it is important to study the effect of organic cosolvent molecules on the structure and dynamics of the enzyme, in particular, the effect of cosolvent molecules on the active site's structure and dynamics. In this paper, we have studied, using molecular dynamics (MD) simulations, the F87A mutant of P450 BM-3 in the presence of DMSO as cosolvent, to understand the role of the F87A substitution for its catalytic activity. This mutant exhibits an altered regioselectivity and substrate specificity compared with wild-type; however, it has lower tolerance toward DMSO. The simulation results offer an explanation for the DMSO sensitivity of the F87A mutant. Our simulation results show that the F87 side chain prevents the disturbance of the water molecule bound to the heme iron by DMSO molecules. The absence of the phenyl ring in F87A mutant promotes interactions of the DMSO molecule with the heme iron resulting in water displacement by DMSO at the catalytic heme center. 相似文献
13.
本研究通过体外生化实验研究细胞色素P450 3A7对维生素D3的羟化作用。根据GenBank报道的序列设计特异引物,扩增cyp3a7的编码区,将cyp3a7的编码区插入到pcDNATM3.1/myc-His(-) A的XhoⅠ/Bam HⅠ,通过测序检测序列的正确性。pcDNA-CYP3A7及pcDNA分别瞬时转染293T细胞,48 h后收集细胞,提取S9组分,用Bradford法测定蛋白质浓度。S9组分经12%SDS-PAGE凝胶电泳和Western blotting检测,用myc抗体作为一抗检测CYP3A7在293T细胞的表达水平。0.6 mg S9组分与1μmol/L维生素D3于37℃孵育30 min,用4倍体积的氯仿甲醇(体积比为3∶1)抽提,有机相在氮气流下吹干,残基用于HPLC分析。结果显示,重组表达CYP3A7的293T细胞的S9组分通过Western blotting检测到了特异的约60 kD的条带,对照样品未检测到特异条带的蛋白质。重组表达CYP3A7的293T细胞S9组分的孵育样品通过HPLC检测到了25-羟基维生素D3,对照样品未检测到25-羟基维生素D3。结果表明重组表达的CYP3A7羟化维生素D3生成25-羟基维生素D3。本研究为进一步探究还有哪些P450参与维生素D3在鸡体内的代谢,为阐明其代谢途径提供理论依据。 相似文献
14.
Escherichia coli BL21, expressing a quintuple mutant of P450BM-3, oxyfunctionalizes α-pinene in an NADPH-dependent reaction to α-pinene oxide, verbenol, and myrtenol. We optimized the whole-cell
biocatalyst by integrating a recombinant intracellular NADPH regeneration system through co-expression of a glucose facilitator
from Zymomonas mobilis for uptake of unphosphorylated glucose and a NADP+-dependent glucose dehydrogenase from Bacillus megaterium that oxidizes glucose to gluconolactone. The engineered strain showed a nine times higher initial α-pinene oxide formation
rate corresponding to a sixfold higher yield of 20 mg g−1 cell dry weight after 1.5 h. The initial total product formation rate was 1,000 μmol h−1 μmol−1 P450 leading to a total of 32 mg oxidized products per gram cell of dry weight after 1.5 h. The physiological functioning
of the heterologous cofactor regeneration system was illustrated by a sevenfold increased α-pinene oxide yield in the presence
of glucose compared to glucose-free conditions. 相似文献
15.
16.
《Process Biochemistry》2014,49(4):655-659
An efficient biocatalytic process for the production of nicotinic acid (niacin) from 3-cyanopyridine was developed using cells of recombinant Escherichia coli JM109 harboring the nitrilase gene from Alcaligenes faecalis MTCC 126. The freely suspended cells of the biocatalyst were found to withstand higher concentrations of the substrate and the product without any signs of substrate inhibition. Immobilization of the cells further enhanced their substrate tolerance, stability and reusability in repetitive cycles of nicotinic acid production. Under optimized conditions (37 °C, 100 mM Tris buffer, pH 7.5) for the immobilized cells, the recombinant biocatalyst achieved a 100% conversion of 1 M 3-cyanopyridine to nicotinic acid within 5 h at a cell mass concentration (fresh weight) of 500 mg/mL. The high substrate/product tolerance and stability of the immobilized whole cell biocatalyst confers its potential industrial use. 相似文献
17.
《Bioscience, biotechnology, and biochemistry》2013,77(9):1922-1927
Actinomycete cytochrome P450 from Nonomuraea recticatena NBRC 14525 (P450moxA) catalyzes the hydroxylation of a broad range of substrates, including fatty acids, steroids, and various aromatic compounds. Hence, the enzyme is potentially useful in medicinal applications, but the activity is insufficient for practical use. Here we applied directed evolution to enhance the activity. A random mutagenesis library was screened using 7-ethoxycoumarin as a substrate to retrieve 17 variants showing >2-fold activities. Twenty-five amino acid substitutions were found in the variants, of which five mutations were identified to have the largest effects (Q87W, T115A, H132L, R191W, and G294D). These mutations additively increased the activity; the quintet mutant had 20-times the activity of the wildtype. These five single mutations also increased in activity toward structurally distinct substrates (diclofenac and naringenin). Based on the three-dimensional structure of the enzyme, we discerned that mutations in the substrate recognition site improved the activity, which was substrate dependent; mutations apart from the active site improved the activity as well as the substrates did. 相似文献
18.
Peña-Rico M Guadalupe Ortiz-López M Camacho-Castillo L Cárdenas M Pedraza-Chaverri J Menjívar M 《Life sciences》2006,79(7):702-708
The nephrotic syndrome is a renal disease characterized by proteinuria, hypoproteinemia, edema and hyperlipidemia. It has been reported that female nephrotic rats are characterized by loss of the oestrus cycle, follicle atresia, low gonadotropin and steroid concentrations; particularly, undetectable estradiol levels. Therefore, to determine the mechanisms involved in the ovarian steroidogenesis impairment, in this present study we evaluated the ovarian expression of the essential steroidogenesis components: cytochrome P450 side cholesterol chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR). The experiments were conducted in the rat experimental model of nephrosis induced by puromycin aminonucleoside (PAN) and in control groups. The evaluation of the expression of P450scc and StAR mRNA were performed during the acute phase of nephrosis as well as after the exogenous administration of 1 or 4 doses of human chorionic gonadotrophin (hCG), or a daily dose of FSH or FSH+hCG for 10 days. In addition, serum hormone concentrations, intra-ovarian steroid content, and the reproductive capacity were determined. The results revealed a decreased expression of mRNA of P450scc enzyme and StAR during nephrosis, and eventhough they increased after gonadotropins treatment, they did not conduce to a normal cycling rat period or fertility recovery. This study demonstrates that the mechanism by which ovarian steroid biosynthesis is altered during acute nephrosis involves damage at the P450scc and StAR mRNA synthesis and processing. 相似文献
19.
The purpose of this study was to identify isozyme-specific antibodies and use them to determine the expression levels of four P450 3A enzymes in the livers of vehicle- and pregnenolone 16alpha-carbonitrile (PCN)-treated rats of both sexes, since previous work on mRNA levels has shown considerable sexual dimorphism. Using Western blot analysis with four isozyme-specific antibodies, we show that P450 3A1, 3A2, and 3A9 were expressed in vehicle-treated adult female rats at very low levels whereas P450 3A18 was not detected. PCN treatment of females strongly induced the expression of P450 3A1 in the livers with protein product increases of 214-, 3-, and 5-fold for P450 3A1, 3A2, and 3A9, respectively, and P450 3A18 was induced to 3.7 pmol/mg protein. In contrast, all four P450 3As were detected in livers of vehicle-treated males, in the order of 3A2 > 3A18 > 3A9 approximately = 3A1. The protein product increases induced by PCN treatment of male rats were 92-, 3-, 6-, and 16-fold for P450 3A1, 3A2, 3A9, and 3A18, respectively. 相似文献
20.
Davydov DR Rumfeldt JA Sineva EV Fernando H Davydova NY Halpert JR 《The Journal of biological chemistry》2012,287(9):6797-6809
The mechanisms of ligand binding and allostery in the major human drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) were explored with fluorescence resonance energy transfer (FRET) using a laser dye, fluorol-7GA (F7GA), as a model substrate. Incorporation into the enzyme of a thiol-reactive FRET probe, pyrene iodoacetamide, allowed us to monitor the binding by FRET from the pyrene donor to the F7GA acceptor. Cooperativity of the interactions detected by FRET indicates that the enzyme possesses at least two F7GA-binding sites that have different FRET efficiencies and are therefore widely separated. To probe spatial localization of these sites, we studied FRET in a series of mutants bearing pyrene iodoacetamide at different positions, and we measured the distances from each of the sites to the donor. Our results demonstrate the presence of a high affinity binding site at the enzyme periphery. Analysis of the set of measured distances complemented with molecular modeling and docking allowed us to pinpoint the most probable peripheral site. It is located in the vicinity of residues 217-220, similar to the position of the progesterone molecule bound at the distal surface of the CYP3A4 in a prior x-ray crystal structure. Peripheral binding of F7GA causes a substantial spin shift and serves as a prerequisite for the binding in the active site. This is the first indication of functionally important ligand binding outside of the active site in cytochromes P450. The findings strongly suggest that the mechanisms of CYP3A4 cooperativity involve a conformational transition triggered by an allosteric ligand. 相似文献