The presence of CYP79 homologues in glucosinolate-producing plants shows evolutionary conservation of the enzymes in the conversion of amino acid to aldoxime in the biosynthesis of cyanogenic glucosides and glucosinolates

A cDNA encoding CYP79B1 has been isolated from Sinapis alba. CYP79B1 from S. alba shows 54% sequence identity and 73% similarity to sorghum CYP79A1 and 95% sequence identity to the Arabidopsis T42902, assigned CYP79B2. The high identity and similarity to sorghum CYP79A1, which catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin, suggests that CYP79B1 similarly catalyses the conversion of amino acid(s) to aldoxime(s) in the biosynthesis of glucosinolates. Within the highly conserved PERF and the heme-binding region of A-type cytochromes, the CYP79 family has unique substitutions that define the family-specific consensus sequences of FXP(E/D)RH and SFSTG(K/R)RGC(A/I)A, respectively. Sequence analysis of PCR products generated with CYP79B subfamily-specific primers identified CYP79B homologues in Tropaeolum majus, Carica papaya, Arabidopsis, Brassica napus and S. alba. The five glucosinolate-producing plants identified a CYP79B amino acid consensus sequence KPERHLNECSEVTLTENDLRFISFSTGKRGC. The unique substitutions in the PERF and the heme-binding domain and the high sequence identity and similarity of CYP79B1, CYP79B2 and CYP79A1, together with the isolation of CYP79B homologues in the distantly related Tropaeolaceae, Caricaceae and Brassicaceae within the Capparales order, show that the initial part of the biosynthetic pathway of glucosinolates and cyanogenic glucosides is catalysed by evolutionarily conserved cytochromes P450. This confirms that the appearance of glucosinolates in Capparales is based on a cyanogen predisposition. Identification of CYP79 homologues in glucosinolate-producing plants provides an important tool for tissue-specific regulation of the level of glucosinolates to improve nutritional value and pest resistance.     

Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin

A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450 – (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG – was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained.Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH–cytochrome P450–reductase in L--dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis. In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile. In vivo administration of oxime to E. coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E. coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction.CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin. Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450–reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin. Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.     

Ester Formation by Alcohol Acetyltransferase from Brewers’ Yeast

Alcohol acetyltransferase responsible for the formation of acetate esters during beer fermentation was found to be localized at the cell membrane of brewers’ yeast. This cell membrane-bound enzyme was purified 120-fold by solubilization with Triton X-100, gel filtration on a Sepharose 6B column and chromatography on a DEAE-Sephadex A-50 column. The enzyme was most active at 30°C at pH 7 ? 8. It was least active against C₃ alcohol among C₁ ? C₆ alcohols, and slightly more active against straight-chain alcohols than against branched-chain alcohols with the same carbon number. The enzyme was strongly inhibited by unsaturated fatty acids, heavy metal ions and sulfhydryl reagents.     

Sunflower cDNA Encoding New Cytochrome P450

The primary structure of the cDNA clone SF28 was determined in sunflower (Helianthus annuusL.) flowers. The clone comprises a 874-bp insert corresponding to 227 amino acid residues of the C-terminal part of the cytochrome P450 gene. The sunflower cytochrome P450 was considerably different from the already known plant and animal cytochromes P450.     

Productive and non-productive complexes in cytochrome P450-containing systems

The equilibrium dissociation constants K_D, the complex association / dissociation rate constants (k _on /k _off) and lifetimes of the complexes of redox partners were measured for three cytochrome P450-containing monooxygenase systems (P450cam, P450scc, and P450 2B4) under hydroxylation conditions. The Q parameter representing the ratio of protein-protein complex lifetime (τ _lT ) to time required for a single hydroxylation cycle (τ_turnover) was introduced for estimation of productivity of complexes formed within the systems studied. The Q parameter was insignificantly changed upon transition from the oxidation to hydroxylation conditions. Lifetimes (τ _lT ) for the binary complexes formed within the P450cam and the P450scc systems obligatory requiring an intermediate electron transfer protein between the reductase and cytochrome P450 could not realize hydroxylation reactions for substrates with known τ_turnover and so they were non-productive while the binary complexes formed within the P450 2B4 system, not requiring such intermediate electron-transfer protein, appeared to be productive. Formation of ternary complexes was demonstrated under hydroxylation conditions in all three systems. Analysis of Q values led to the conclusion that the ternary complexes formed within the P450cam and the P450scc systems were productive. In the case of the P450 2B4 system, more than half (about 60%) ternary complexes were also found to be productive.     

细胞色素P450基因及其在植物改良中的应用

细胞色素P450是一类含血红素的氧化还原酶类,它参与多种生化反应,在防御生物免受病虫害及逆境胁迫等方面具有重要作用。生物基因组序列分析表明,它是一个基因超家族。许多细胞色素P450基因已被鉴定和克隆,并应用于植物遗传改良;在转基因培育多抗性植物、创造植物雄性不育系,提高植物降解化学农药残留等污染物的能力和有效生产具有药用价值的化合物等方面已取得可喜进展,显示出广阔的应用前景。Abstract:Cytochrome P450s are heme-containing mixed-function oxidases,involving in lots of biochemical reactions.They play an important role in preventing plants from pathogen and insect attacks and environmental stress.Sequence analysis of genomes has revealed that P450 is a gene super-family.Many cytochrome P450s have been characterized and cloned.Some of them have been used in plant genetic improvement.A great progress has been made in using these P450 genes to create the transgenic plants with multiple resistances,male sterility,higher capability to dissolve toxic chemicals and pollutants and effective productivity of high valuable compounds,indicating P450 genes have a broad prospect with great potential application.     

Expression analysis of the mixed function oxidase system in rat brain by the polymerase chain reaction

Metabolism of therapeutic drugs in the body by the mixed function oxidase system is an important consideration in the analysis of a drug's effectiveness. P450-dependent metabolism within the brain of a neuro-specific drug may affect the drug's course of action. To determine whether cytochrome P450 was expressed in brain, RNA was isolated from the whole brains of rats treated with a variety of known hepatic P450 inducers, including amitriptyline, imipramine, isosafrole, phenobarbital, and -naphthoflavone. The RNA was analyzed for the presence of P450 isozymes by the PCR technique. Differential expression of P450IA1, P450IIB1, P450IIB2, P450IID, and P450IIE1 was detected in the brain samples, depending on the treatment. Cytochrome P450 reductase expression was also detected in the brain samples, giving strong evidence that the brain contains a competent mixed function oxidase system under all conditions studied. (Mol Cell Biochem120: 171–179, 1993)Thesis student of the Graduate School of Biomedical Sciences, the University of Texas Health Science Center at Houston     

木薯Mlo基因家族成员的鉴定及其序列特征分析

M／o基因家族是植物重要的抗病基因。本文通过系统分析木薯基因组数据库,从中共鉴定出21个M／o成员,其中20个具有完整序列,1个只有部分序列。对其中20个具有完整序列的基因与其他物种的Mlo基因进行聚类关系分析,结果显示,可将木薯Mlo基因家族分为6类（I～VI）,其中4类都包括有来自拟南芥的Mlo基因,第vI类只包括2个木薯Mlo基因,可能是木薯中特有的一类Mlo;6个木薯Mlo与已知的抗病Mlo基因分别聚在第1V和第V类,这6个基因可能是木薯基因组中具有抗病功能的Mlo。对所有的木薯Mlo蛋白进行结构分析发现,除了MeMl020外,其他蛋白均具有6～8个跨膜结构,其中3个蛋白具有N端信号肽。     

Preparation of Cell Wall Fractions by Various Ways and Activity of Bound Saccharase in Sugar Beet Seedlings

Washed cells of facultative methylotrophs which have the serine pathway showed high activities for l-methionine formation from dl-homocysteine, in the presence of methanol as methyl donor. Strain FM 518, isolated from soil and identified as a bacterium belonging to the genus Pseudomonas, showed the highest activity for l-methionine formation and was used as the parental strain for breeding the l-methionine-producing mutants. An ethionine-resistant mutant, FE 244, derived from strain FM 518, accumulated 0.8 mg/ml l-methionine in a methanol-medium under optimum conditions.     

Structural analysis of CYP2R1 in complex with vitamin D3

The activation of vitamin D to its hormonal form is mediated by cytochrome P450 enzymes. CYP2R1 catalyzes the initial step converting vitamin D into 25-hydroxyvitamin D. A CYP2R1 gene mutation causes an inherited form of rickets due to 25-hydroxylase deficiency. To understand the narrow substrate specificity of CYP2R1 we obtained the hemeprotein in a highly purified state, confirmed the enzyme as a vitamin D 25-hydroxylase, and solved the crystal structure of CYP2R1 in complex with vitamin D₃. The CYP2R1 structure adopts a closed conformation with the substrate access channel being covered by the ordered B′-helix and slightly opened to the surface, which defines the substrate entrance point. The active site is lined by conserved, mostly hydrophobic residues. Vitamin D₃ is bound in an elongated conformation with the aliphatic side-chain pointing toward the heme. The structure reveals the secosteroid binding mode in an extended active site and allows rationalization of the molecular basis of the inherited rickets associated with CYP2R1.     

木薯的组织培养和基因转化

木薯是重要的热带作物之一, 其块根富含淀粉, 是热带、亚热带地区近５ 亿人粮食的主要来源。简单介绍近年来运用生物技术改良木薯的研究进展。     

植物细胞色素P450

对植物细胞色素P450(CYP450)基因的分离,植物CYP450在苯丙烷类物质、芥子油苷及IAA和萜类等物质的生物合成中的功能,以及对天然生物合成与人工合成物质的解毒功能等研究进展作了简要的综述。指出分离植物细胞色素P450基因,并对其生物学功能进行分析以及植物细胞色素P450降解除草剂的机制及其在环境生物修复等方面的应用是今后一段时间内植物CYP450领域的研究热点。     

Organization of the Biosynthetic Gene Cluster for the Polyketide Antitumor Macrolide,Pladienolide, in Streptomyces platensis Mer-11107

Pladienolides are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107. They show strong antitumor activity and are a potential lead in the search for novel antitumor agents. We sequenced the 65-kb region covering the biosynthetic gene cluster, and found four polyketide synthase genes (pldAI-pldAIV) composed of 11 modules, three genes involved in post-modifications (pldB-D), and a luxR-family regulatory gene (pldR). The thioesterase domain of pldAIV was more dissimilar to that of polyketide synthase systems synthesizing 12/14-membered macrolide polyketides than to that of systems synthesizing other cyclic polyketides. The pldB gene was identified as a 6-hydroxylase belonging to a cytochrome P450 of the CYP107 family. This was clarified by a disruption experiment on pldB, in which the disruptant produced 6-dehydroxy pladienolide B. Two genes located downstream of pldB, designated pldC and pldD, are thought to be a probable genes for 7-O-acetylase and 18, 19-epoxydase respectively.     

Preliminary crystallographic analysis of an enzyme involved in erythromycin biosynthesis: Cytochrome P450eryF

Cytochrome P450eryF was overexpressed in Escherichia coli and purified in high yield. Crystals of the protein in the presence of the substrate, 6-deoxyerythronolide B, have been obtained by the hanging drop vapor diffusion method, using polyethylene glycol 4000 as a precipitant. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 54.16 Å, b = 79.67 Å, and c = 99.48 Å and one molecule per asymmetric unit. A complete native data set has been collected to a resolution of 2.1 Å, and anomalous dispersion difference Patterson maps have revealed the location of the single heme iron atom. © 1994 Wiley-Liss, Inc.     

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。