P450酶系中高效电子传递链的构建及应用

细胞色素P450作为单加氧酶的主要成员,能够在多种化合物中引入氧分子,催化包括羟化在内的多种反应。在级联的氧化还原反应中,需特定的电子传递链将氧原子中的电子传递至P450单加氧酶的亚铁红素结构中,并最终催化底物氧化,而电子传递体系的低效性往往成为整个反应的限速步骤。本文在介绍P450单加氧酶电子传递链基本结构的基础上,着重阐述对于细胞色素P450酶系中电子传递链未知或者内源性电子传递效率较低的情况下,利用DNA重组技术构建高效的电子传递链从而提高P450单加氧酶的催化效率相关研究进展,主要从细菌及真核细胞线粒体电子传递链(ClassⅠ)及真核生物细胞色素C还原酶CPR(ClassⅡ),天然融合电子传递链及人工融合蛋白电子传递链的构建及其应用展开。     

云芝NADPH-细胞色素P450还原酶在大肠杆菌中表达及酶学特性分析

云芝Trametes versicolor具有很强的环境有机污染物降解能力,其烟酰胺腺嘌呤二核苷酸-细胞色素P450还原酶(NADPH-cytochrome P450 reductase,CPR)为细胞色素P450酶(Cytochrome P450s,CYPs)提供电子,参与有机污染物的降解过程。序列分析显示,云芝基因组拥有1个潜在CPR序列和多个潜在CYP序列。为深入研究云芝CPR参与细胞降解有机污染物的分子机制,实验进行了云芝CPR在大肠杆菌中异源表达和酶学特性分析。结果表明,经IPTG诱导后,去除预测的N端膜锚定区域(氨基酸残基1–24)的CPR蛋白(CPRΔ24)可在重组菌中实现可溶性表达,且表达蛋白的分子量与理论值(78 kDa)一致。镍离子亲和层析和分子筛层析纯化后测得其比活性为5.82 U/mg。酶学性质分析显示,重组CPRΔ24的最适温度和pH分别为35℃和8.0,并对一些金属离子及有机溶剂具有不同程度的耐受性。酶在35℃、pH 8.0反应条件下对NADPH的动力学参数K_m和k_(cat)分别为19.7μmol/L、3.31/s;对底物细胞色素c的动力学参数K_m和k_(cat)分别为25.9μmol/L、10.2/s。以上研究为探究云芝CPR在环境有机污染物降解途径中的功能机制奠定基础。     

鸡细胞色素P450 1A5的原核表达与活性重组

旨在对鸡细胞色素P450 1A5(CYP1A5)蛋白进行体外功能研究,采用大肠杆菌系统进行CYP1A5的异源表达。以鸡的cDNA为模板,扩增出CYP1A5基因,将该基因的N端编码区进行修饰,并连接到pCW载体中构建His-CYP1A5,经IPTG诱导在大肠杆菌中表达。经CO-差示光谱检测,所获得的His-CYP1A5具有典型的P450吸收峰。该蛋白与细胞色素P450还原酶(CPR)进行体外重组,构成的重组酶系表现出乙氧基试卤灵-O-脱乙基酶活性。结果表明,所采用的表达策略可以成功产生出具有催化活性的鸡细胞色素P450 1A5(CYP1A5)蛋白。     

在大肠杆菌中拓展合成途径合成异胡椒醇北大核心

天然异胡椒醇作为一种植物来源香料,具有很高的经济价值。目前国内外还未见利用大肠杆菌工程菌株转化生产异胡椒醇的报道。【目的】构建工程菌,采用生物合成方法获得天然香料异胡椒醇。【方法】对来自胡椒薄荷的细胞色素P450家族的柠檬烯-3-羟化酶(LIM3H)基因PM2进行改造,截短LIM3H的N端疏水区,分别添加17α短肽或2B1短肽增加其亲水性,采用CO差示光谱法检测LIM3H活性;将N端疏水区截短后的来自拟南芥(Arabidopsis thaliana)的NADPH-细胞色素P450还原酶基因(trATR)和修饰后LIM3H基因(Modi-LIM3H)融合后表达,最终以柠檬烯为底物进行全细胞转化。【结果】3种N端修饰LIM3H基因均检测到LIM3H表达和异胡椒醇生成,其中17α短肽修饰后的蛋白表达量最高,异胡椒醇产量达到1.94 mg/L。【结论】本研究在大肠杆菌中增加外源LIM3H与ATR,成功催化柠檬烯生成异胡椒醇,为天然异胡椒醇的生产提供了新的方法。     

猪流行性腹泻病毒S2截短肽的原核表达及其线性B细胞表位区鉴定

【背景】由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)引起的猪流行性腹泻给养猪业造成了巨大的经济损失。PEDV S蛋白可以诱导宿主产生中和抗体。【目的】原核表达PEDV CV777疫苗株S2截短肽(aa:961-1 382)并制备其多克隆抗体;鉴定表达的S2截短肽上的线性B细胞表位区。【方法】将经密码子优化的PEDV S2截短肽编码DNA (s2t)克隆至载体p ET-28a中并转化Escherichia coli BL21(DE3),利用IPTG诱导S2截短肽表达。以经SDS-PAGE切胶纯化的重组S2截短肽免疫新西兰大白兔制备多克隆抗体。在E. coli BL21中GST融合表达覆盖S2截短肽序列全长、彼此重叠8个氨基酸残基的系列16肽。以制备的抗S2截短肽兔血清为一抗,通过Westernblot(WB)筛选系列16肽中的阳性反应性16肽,鉴定S2截短肽上的线性B细胞表位区。【结果】重组PEDV S2截短肽的相对分子质量约为50 kD;诱导4 h表达量最高,且主要形成包涵体。WB结果显示,纯化的S2截短肽能被猪抗PEDV血清识别;以纯化的S2截短肽免疫新西兰大白兔制备多抗血清,ELISA法检测抗体效价位于1:25 600-1:102 400之间。免疫组化和间接免疫荧光分析均表明,制备的多抗血清可以识别Vero细胞培养的PEDV DR13弱毒株。以制备的多抗血清通过WB从52个GST融合表达的16肽中鉴定到11个阳性反应性16肽。WB分析显示,得到的阳性反应性16肽都可以被猪抗PEDV血清识别。鉴定到的阳性16肽在S2截短肽上形成4个线性B细胞表位区(aa:969-984;1 065-1 096;1 225-1 280;1 361-1 382)。【结论】高效价抗PEDV S2截短肽多克隆抗体的制备和S2截短肽上线性B细胞抗原表位区的确定有助于了解S蛋白的结构与功能,为建立有效的PEDV检测方法奠定了基础。     

细胞色素P450介导的昆虫抗药性

本文介绍了昆虫细胞色素P450(简称P450)及其介导抗性的分子基础的研究进展。细胞色素：P450在转录水平上的过量表达是P450介导抗性的主要机制,P450的氨基酸残基改变也可能改变昆虫的抗药性。     

斑点叉尾鮰(Ictalurus punctatus)LEAP2成熟肽在大肠杆菌中的融合表达

以斑点叉尾鮰(Ictalurus punctatus)肝脏为基因克隆的材料,通过RT-PCR对编码LEAP2成熟肽区域41个氨基酸的cDNA片段(mLEAP2)进行克隆。根据斑点叉尾鮰与其他鱼类、哺乳动物及两栖动物等其他物种LEAP2成熟肽区域氨基酸序列的比对结果,发现存在14个保守的氨基酸残基,其中4个高度保守的半胱氨酸残基位于成熟肽的羧基端区域,在空间上可形成两对二硫键,推测与LEAP2的抗菌活性有关。进一步构建重组表达质粒pET32a-mLEAP2,使mLEAP2基因与携带有6×His-tag标签和肠激酶识别位点的硫氧还蛋白trxA基因融合,转化至大肠杆菌BL21(DE3)后,在25℃下,经0.7 mmol/L IPTG诱导培养16 h后,成功表达了trxA-mLEAP2融合蛋白。Tricine-SDS-PAGE显示,细胞经超声破碎后,上清和沉淀中均含融合蛋白,采用固化金属离子亲和层析(IMAC)对其进行纯化,得到了纯化的融合蛋白。     

东亚飞蝗细胞色素P450基因的克隆及表达分析

本文克隆了东亚飞蝗Locusta migratoria manilensis(Meyen)细胞色素P450(cytochrome P450)基因全长,表达重组蛋白,并对其可溶性进行了分析。通过提取东亚飞蝗总的RNA,反转录成cDNA,设计特异性引物,PCR克隆东亚飞蝗细胞色素P450基因,将测序正确的目的片段克隆至原核表达载体pET-28a中,在大肠埃希菌Escherichia coli Rosetta中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测重组蛋白表达结果。结果表明:东亚飞蝗细胞色素P450基因开放阅读框全长为1 551 bp,编码516个氨基酸,与GenBank中已登录的东亚飞蝗细胞色素P450基因(HM153426)的同源性为99%,重组质粒pET-28a-P450在E.coli Rosetta中获得高效表达,重组蛋白相对分子质量(Mr)约为53 000,主要以包涵体的形式存在。     

冬虫夏草菌NADPH-细胞色素P450还原酶基因的生物信息学分析

细胞色素P450家族和真菌致病性有关,而细胞色素P450的催化活性又依赖于烟酰胺腺嘌呤二核苷酸磷酸(NADPH)-细胞色素P450还原酶(CPR)提供的电子.为进一步探讨NADPH-CPR基因多样性对冬虫夏草菌Ophiocordyceps sinensis侵染寄主的影响,扩增获得2个冬虫夏草菌NADPH-CPR基因,利用生物信息学方法分析冬虫夏草菌NADPH-CPR基因序列特征及预测编码蛋白的理化性质和结构特征等.结果表明:2个冬虫夏草菌NADPH-CPR基因的核苷酸序列长度分别为2 192 bp和3 603 bp,编码692和1 065个氨基酸,对应3个外显子和7个外显子.2个冬虫夏草菌NADPH-CPR基因编码的蛋白均为亲水性蛋白,最可能分别定位于内质网的分泌途径和细胞质上,蛋白质二级结构原件为α螺旋、无规则卷曲、延伸链、β-转角,其中NADPH-CPR-1的α螺旋占比小于NADPH-CPR-2,而无规则卷曲和延伸链占比则大于NADPH-CPR-2.2个冬虫夏草菌NA4DPH-CPR基因编码的蛋白均具有NADPH-CPR典型的结构域,而NADPH-CPR-2在N端多了1个细胞色素P450结构域.     

大肠杆菌YggG与结合蛋白Era的相互作用研究

Era是细菌生长必须的一高度保守的GTPase。yggG是从大肠杆菌全基因组文库中钓取并克隆的Era结合蛋白基因,进一步的研究表明该基因在大肠杆菌中的表达与环境应激相关,提示yggG基因产物参与细菌的应激调控。为了阐明YggG蛋白与Era蛋白间的相互关系,利用所构建的双启动子表达载体pDH2-YggG-Ptac-Era在同一细胞中同时表达YggG与Era蛋白,并通过免疫共沉淀实验检测细菌裂解产物YggG与Era蛋白间的相互作用;在此基础上,构建并表达纯化了GST融合的Era蛋白氨基端截短肽和Era羧基端截短肽,通过GST Pull-down检测了Era不同功能区域与YggG蛋白间的相互作用。结果显示, Era/YggG 复合物仅存在于同时过表达Era和YggG蛋白的细菌细胞内,不诱导Era或者不诱导YggG蛋白过表达,均检测不到Era/YggG 复合物存在;纯化的GST不能Pull-down YggG蛋白,而纯化的GST融合的Era蛋白、Era氨基端截短肽及Era羧基端截短肽均可以Pull-down YggG蛋白;GST融合Era氨基端截短肽和GST融合的Era蛋白对YggG蛋白结合作用明显高于GST融合的Era蛋白羧基端截短肽。上述结果说明,YggG是一大肠杆菌Era结合蛋白,YggG与Era的氨基端和羧基端的结合活性存在差异。     

Role of C-terminal sequence of cytochrome P450scc in folding and functional activity

To elucidate the role of Arg472 and C-terminal sequence of the mature form of cytochrome P450scc, a mitochondrial cytochrome P450, in the present work we have performed sequential removal of the C-terminal amino acid residues of the hemeprotein and evaluated their functional role in folding and catalysis. The removal of 2, 4, 7, or 9 amino acid residues (cytochrome P450scc mutants Delta2, Delta4, Delta7, and Delta9) does not significantly affect the physicochemical properties of the truncated forms of cytochrome P450scc, but results in significant increase in the expression level of the hemeprotein in Escherichia coli (Delta4 cytochrome P450scc mutant). However, removal of 10 C-terminal amino acid residues (Delta10 cytochrome P450scc) of mature form of cytochrome P450scc (replacement of codon for Arg472 for stop-codon) is followed by loss of the ability for correct folding in E. coli. Based on these data, it is concluded that the C-terminal amino acid residues of cytochrome P450scc (DeltaArg472-Ala481) play an important role in correct recombinant protein folding and heme binding by cytochrome P450scc during its expression in E. coli, while folding of mitochondrial cytochrome P450scc during its heterologous expression in bacterial cells is more similar to the folding of prokaryotic soluble cytochrome P450's than to microsomal cytochrome P450's.     

Molecular cloning of monkey liver cytochrome P-450 cDNAs: similarity of the primary sequences to human cytochromes P-450.

Three cDNAs coding for monkey cytochrome P-450 (P450) 2C, 2E and 3A (MKmp13, MKj1 and MKnf2, respectively) were isolated from a lambda gt11 cDNA library of a liver from a 3-methylcholanthrene (3MC)-treated crab-eating monkey, using cDNA fragments for human P450 2C, 2E and 3A as respective probes. MKmp13 and MKnf2 were 1901 and 2032 bp long, containing entire coding regions for polypeptides of 490 and 503 residues, respectively. The deduced N-terminal amino acid sequences of MKmp13 and MKnf2 were identical with those of P450-MK1 and P450-MK2, which had been purified from liver microsomes of untreated and polychlorinated biphenyl (PCB)-treated crab-eating monkeys, respectively. MKj1 was 1508 bp long, encoding a polypeptide of 449 residues, which is presumed to lack N-terminal 45 residues as compared with the sequence for human P450 2E1. Northern blot analysis indicated that monkey P450 2C, 2E and 3A mRNAs were expressed constitutively in monkey livers. P450 2E and 3A mRNAs were induced by both 3MC and PCB, while P450 2C mRNA was induced only by PCB. The deduced amino acid sequences of four monkey cytochrome P-450 cDNAs, including P450 1A1 (MKah1) which we isolated previously, were more than 92% identical with those of corresponding human cytochrome P-450 cDNAs.     

An enzymatically active chimeric protein containing the hydrophilic form of NADPH-cytochrome P450 reductase fused to the membrane-binding domain of cytochrome b5.

The microsomal flavoprotein NADPH-cytochrome P450 reductase (CPR) contains an N-terminal hydrophobic membrane-binding domain required for reconstitution of hydroxylation activities with cytochrome P450s. In contrast, cytochrome b5 (b5) contains a C-terminal hydrophobic membrane-binding domain required for interaction with P450s. We have constructed, expressed and purified a chimeric flavoprotein (hdb5-CPR) where the C-terminal 45 amino acid residues of b5 have replaced the N-terminal 56 amino acid domain of CPR. This hybrid flavoprotein retains the catalytic properties of the native CPR and is able to reconstitute fatty acid and steroid hydroxylation activities with CYP4A1 and CYP17A. However hdb5-CPR is much less effective than CPR for reconstituting activity with CYP3A4. We conclude that differences on the surface of the P450s reflect unique and specific information essential for the recognition needed to establish reactions of intermolecular electron transfer from the flavoprotein CPR.     

Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli

We have expressed in Escherichia coli a cDNA encoding rabbit liver cytochrome P-450IIE1, the ethanol-inducible P-450. The expressed P-450 is located primarily in the bacterial inner cell membrane and comprises 3% of the E. coli total membrane protein. The partially purified cytochrome exhibits a reduced CO difference spectrum with a maximum at 452 nm, characteristic of P-450IIE1, and solubilized membranes or partially purified P-450 preparations reconstituted with NADPH-cytochrome P-450 reductase and phosphatidylcholine catalyze the deethylation of N-nitrosodiethylamine with a turnover number equal to that of purified liver P-450IIE1 (approximately 4.5 nmol/min/nmol of P-450). A modified IIE1 cDNA that encodes a protein lacking amino acids 3-29, a proposed membrane anchor for cytochrome P-450, was also expressed in E. coli and, unexpectedly, the shortened protein was also found to be predominantly located in the bacterial inner membrane rather than the cytosol. Like the full-length protein, this truncated cytochrome has a reduced CO difference spectrum characteristic of P-450IIE1 and is fully active in the deethylation of N-nitrosodiethylamine. These results demonstrate that the NH2-terminal hydrophobic segment is not solely responsible for attachment to the membrane and evidently is not required for proper protein folding or catalytic activity.     

Engineering of proteolytically stable NADPH-cytochrome P450 reductase

NADPH-cytochrome P450 reductase (CPR) is a membrane-bound flavoprotein that interacts with the membrane via its N-terminal hydrophobic sequence (residues 1-56). CPR is the main electron transfer component of hydroxylation reactions catalyzed by microsomal cytochrome P450s. The membrane-bound hydrophobic domain of NADPH-cytochrome P450 reductase is easily removed during limited proteolysis and is the subject of spontaneous digestion of membrane-binding fragment at the site Lys56-Ile57 by intracellular trypsin-like proteases that makes the flavoprotein very unstable during purification or expression in E. coli. The removal of the N-terminal hydrophobic sequence of NADPH-cytochrome P450 reductase results in loss of the ability of the flavoprotein to interact and transfer electrons to cytochrome P450. In the present work, by replacement of the lysine residue (Lys56) with Gln using site directed mutagenesis, we prepared the full-length flavoprotein mutant Lys56Gln stable to spontaneous proteolysis but possessing spectral and catalytic properties of the wild type flavoprotein. Limited proteolysis with trypsin and protease from Staphylococcus aureus of highly purified and membrane-bound Lys56Gln mutant of the flavoprotein as well as wild type NADPH-cytochrome P450 reductase allowed localization of some amino acids of the linker fragment of NADPH-cytochrome P450 reductase relative to the membrane. During prolong incubation or with increased trypsin ratio, the mutant form showed an alternative limited proteolysis pattern, indicating the partial accessibility of another site. Nevertheless, the membrane-bound mutant form is stable to trypsinolysis. Truncated forms of the flavoprotein (residues 46-676 of the mutant or 57-676 of wild type NADPH-cytochrome P450 reductase) are unable to transfer electrons to cytochrome P450c17 or P4503A4, confirming the importance of the N-terminal sequence for catalysis. Based on the results obtained in the present work, we suggest a scheme of structural topology of the N-terminal hydrophobic sequence of NADPH-cytochrome P450 reductase in the membrane.     

A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.

The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.     

Identification and characterization of an NADPH-cytochrome P450 reductase derived peptide involved in binding to cytochrome P450

The amino acids of cytochrome P450 reductase involved in the interaction with cytochrome P450 were identified with a differential labeling technique. The water-soluble carbodiimide EDC (1-ethyl-3-[3- (dimethylamino)propyl]-carbodiimide) was used with the nucleophile methylamine to modify carboxyl residues. When the modification was performed in the presence of cytochrome P450, there was no inhibition in the ability of the modified reductase to bind to cytochrome P450. However, subsequent modification of the reductase in the absence of cytochrome P450 caused a fourfold increase in the Km and an 80% decrease in kcat/Km (relative to the reductase modified in the first step), for the interaction with cytochrome P450. These effects are attributed to the modification of approximately 3.2 mol of carboxyl residues per mole of reductase. Tryptic peptides generated from the modified reductase were purified by reverse phase high-performance liquid chromatography and characterized. Amino acid sequencing and analysis suggest that the peptide which contains approximately 40% of the labeled carboxyl residues corresponds to amino acid residues 109-130 of rat liver NADPH-cytochrome P450 reductase. One or more of the seven carboxyl containing amino acids within this peptide is presumably involved in the interaction with cytochrome P450.     

Expression, purification and characterization of cytochrome P450 Biol: a novel P450 involved in biotin synthesis in Bacillus subtilis.

The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.     

CYP79B1 from Sinapis alba converts tryptophan to indole-3-acetaldoxime

The cytochrome P450 CYP79B1 from Sinapis alba has been heterologously expressed in Escherichia coli and shown to catalyze the conversion of tryptophan to indole-3-acetaldoxime. Three expression constructs were made, one expressing the native protein and two expressing proteins with different N-terminal modifications. The native construct gave the highest yield as estimated by enzymatic activity per liter of culture. Spheroplasts of E. coli expressing CYP79B1 were reconstituted with the Arabidopsis thaliana NADPH:cytochrome P450 reductase ATR1 heterologously expressed in E. coli to obtain enzymatic activity. This indicates that the E. coli electron-donating system, flavodoxin/flavodoxin reductase, does not support CYP79B1 activity. Recombinant CYP79B1 has a K(m) for tryptophan of 29+/-2 microM and a V(max) of 36.5+/-0.7nmolh(-1)(mlculture)(-1). The identity at the amino acid level of CYP79B1 is, respectively, 93 and 84% to CYP79B2 and CYP79B3 from A. thaliana, and 96% to CYP79B5 (Accession No. AF453287) from Brassica napus. The CYP79B subfamily of cytochromes P450 is likely to constitute a group of orthologous genes in the biosynthesis of indole glucosinolates.     

Molecular cloning and sequence analysis of cDNA coding for rat liver hemoprotein H-450

cDNA clones coding for hemoprotein H-450 were isolated from a rat liver cDNA library using anti-H-450 antibody. The molecular weight calculated from the deduced amino acid sequence comprising 547 amino acid residues was 60,085. The N-terminal sequence and a partial internal amino acid sequence of purified H-450, which were determined chemically, were both found in the amino acid sequence of H-450 deduced from the nucleotide sequence. H-450 mRNA is expressed in liver, kidney, and brain. A homology search of amino acid sequences indicated that H-450 shows no homology with cytochrome P-450, but shows significant homology with bacterial O-acetylserine (thiol)-lyases. However, H-450 has no O-acetylserine (thiol)-lyase activity.