生长素合成途径的研究进展

生长素是一类含有一个不饱和芳香族环和一个乙酸侧链的内源激素, 参与植物生长发育的许多过程。植物和一些侵染植物的病原微生物都可以通过改变生长素的合成来调节植株的生长。吲哚-3-乙酸(IAA)是天然植物生长素的主要活性成分。近年来, 随着IAA生物合成过程中一些关键调控基因的克隆和功能分析, 人们对IAA的生物合成途径有了更加深入的认识。IAA的生物合成有依赖色氨酸和非依赖色氨酸两条途径。依据IAA合成的中间产物不同, 依赖色氨酸的生物合成过程通常又划分成4条支路: 吲哚乙醛肟途径、吲哚丙酮酸途径、色胺途径和吲哚乙酰胺途径。该文综述了近几年在IAA生物合成方面取得的新进展。     

微生物来源天然产物中的β-甲基色氨酸单元及其生物合成研究进展

微生物在次级代谢过程中通常会产生结构复杂、活性多样的天然产物。这些天然产物是新药发展的基础,亦可作为先导化合物或重要的药效基团用于药物研发。结构多样的氨基酸单元是参与合成复杂多样天然产物的重要前体。天然产物中的β-甲基氨基酸单元不仅可以赋予其生物活性,还能增强其生物稳定性而不被肽酶水解。本文综述了含有β-甲基氨基酸单元的天然产物,尤其对含有β-甲基色氨酸单元的天然产物生物合成途径进行了阐释。对β-甲基色氨酸单元生物合成途径的理解结合基因组数据有助于进行新结构天然产物的挖掘,并为运用代谢科学理念和合成生物学技术开发含有该单元的新化合物提供理论基础和可操作遗传元件。     

微生物来源天然产物中的β-甲基色氨酸单元及其生物合成研究进展

微生物在次级代谢过程中通常会产生结构复杂、活性多样的天然产物。这些天然产物是新药发展的基础,亦可作为先导化合物或重要的药效基团用于药物研发。结构多样的氨基酸单元是参与合成复杂多样天然产物的重要前体。天然产物中的β-甲基氨基酸单元不仅可以赋予其生物活性,还能增强其生物稳定性而不被肽酶水解。本文综述了含有β-甲基氨基酸单元的天然产物,尤其对含有β-甲基色氨酸单元的天然产物生物合成途径进行了阐释。对β-甲基色氨酸单元生物合成途径的理解结合基因组数据有助于进行新结构天然产物的挖掘,并为运用代谢科学理念和合成生物学技术开发含有该单元的新化合物提供理论基础和可操作遗传元件。     

红树林放线菌及其天然产物研究进展北大核心CSCD

红树林是热带亚热带潮间带的木本植物群落。为从海洋环境寻找新的天然产物,红树林已经成为放线菌资源收集、天然产物分离鉴定及其生物合成机制研究的热点。从巴哈马红树林分离的盐孢菌所产生的盐孢菌素(Salinosporamide A)已经进入临床研究。从红树林分离的放线菌类群已经达到8个亚目11科24属,并发现新属3个,新种31个。从红树林放线菌分离的新天然产物包括生物碱、喹啉等芳香类、阿扎霉素等大环类脂及吲哚衍生物等,大多数天然产物来源于红树林链霉菌。新型吲哚咔唑、吲哚倍半萜及厦霉素等以新颖的结构备受关注,相关生物合成途径已被揭示。     

COI1参与茉莉酸调控拟南芥吲哚族芥子油苷生物合成过程

芥子油苷是一类具有防御作用的植物次生代谢产物,外源激素茉莉酸对吲哚族芥子油苷的合成具有强烈的诱导作用,但茉莉酸调控吲哚族芥子油苷生物合成的分子机制并不清楚。以模式植物拟南芥(Arabidopsis thaliana)的野生型和coi1-22、coi1-23两种突变体为研究材料,通过茉莉酸甲酯(MeJA)处理,比较了拟南芥野生型和coi1突变体植株吲哚族芥子油苷含量、吲哚族芥子油苷合成前体色氨酸的生物合成基因(ASA1、TSA1和TSB1)、吲哚族芥子油苷生物合成基因(CYP79B2、CYP79B3和CYP83B1)及调控基因(MYB34和MYB51)的表达对MeJA的响应差异,由此确定茉莉酸信号通过COI1蛋白调控吲哚族芥子油苷生物合成,即茉莉酸信号通过信号开关COI1蛋白作用于转录因子MYB34和MYB51,进而调控吲哚族芥子油苷合成基因CYP79B2、CYP79B3、CYP83B1和前体色氨酸的合成基因ASA1、TSA1、TSB1。并且推断,COI1功能缺失后,茉莉酸信号可能通过其他未知调控因子或调控途径激活MYB34转录因子从而调控下游基因表达。     

萘醌-氧吲哚生物碱coprisidins生物合成途径的研究（英文）

【目的】新颖结构的天然萘醌-氧吲哚类生物碱coprisidins(A和B)分离自昆虫肠道相关链霉菌,具有预防癌症的活性。作为首例具有萘醌-氧吲哚骨架的生物碱,对其独特生物合成机理的研究可为Ⅱ型聚酮类化合物生物合成途径提供新的认知。【方法】本研究对coprisidins的产生菌Streptomycessp.SNU607进行全基因组测序,并根据测序结果的生物信息学分析初步定位coprisidins的生物合成基因簇;通过基因敲除以及异源表达手段确定coprisidins的生物合成基因簇;基于体内遗传学实验与生物信息学分析初步推导coprisidins的生物合成途径。【结果】Streptomyces sp. SNU607中有23个基因簇可能参与次级代谢,其中4个基因簇与聚酮合酶(PKS)相关;通过基因敲除与异源表达实验,本研究证实1个Ⅱ型PKS负责coprisidins的生物合成;基于生物信息学分析,我们推测copH/I/M/O/N构成了1个基因盒,并负责起始单元丁酰CoA的合成;KSβ(Cop B)的序列比对表明coprisidins的Ⅱ型PKS系统更倾向于合成C20的初始聚酮链。【结论】Coprisidins的萘醌-吲哚结构是由Ⅱ型PKSs催化形成,我们推测丁酰Co A是coprisidins聚酮骨架的起始单元,在最小PKS、聚酮酶、环化酶的催化下先形成类似蒽环的四环系统,随后在后修饰酶与氧化重排的作用下生成萘醌-氧吲哚骨架。本研究为进一步探究萘醌-氧吲哚类生物碱的生物合成机制奠定了基础,同时增加了Ⅱ型PKSs合成产物的结构多样性。     

外源色氨酸对海水胁迫下长春花幼苗生长及生物碱含量的影响

以长春花[Catharanthus roseus(L.)G.Don]为材料采用温室盆栽法,研究了不同浓度色氨酸对不同浓度海水处理14 d后长春花幼苗生长及吲哚生物总碱含量的影响.结果显示:(1)20%海水中加入不同浓度的色氨酸,长春花幼苗生长受到显著抑制,而丙二醛(MDA)含量、可溶性糖含量、色氨酸脱羧酶(TDC)活性、吲哚生物总碱含量均显著增加;(2)40%海水中加入不同浓度的色氨酸,TDC活性、吲哚生物总碱含量也得到显著提高,但幼苗生长受到严重伤害,生物产量显著降低,吲哚生物总碱的产量太低.研究表明,外源色氨酸能显著提高海水胁迫下长春花吲哚生物总碱的含量,而且用20%的海水中加入500 mg/L的色氨酸最有利于生物碱的积累.     

萘乙酸、激动素和伤害对绿豆子叶愈伤组织形成的作用以及和色氨酸、吲(口朶)乙酸生物合成的关系

本文以绿豆子叶为材料研究了切伤、外源萘乙酸及激动素诱导形成愈伤组织的作用及其与内源色氨酸和吲哚乙酸生物合成的关系。实验结果表明,切伤对于愈伤组织的形成具有重要作用,切伤面积的大小与愈伤组织的增殖成正比。在绿豆子叶愈伤组织形成的初期,游离色氨酸和内源吲哚乙酸的水平均降低,而在后期,组织内部游离色氨酸和吲哚乙酸的含量都有增加。在培养基中加入外源色氨酸可以部分代替萘乙酸促进愈伤组织的形成。可以认为,外源激素诱导愈伤组织的形成是通过促进内源色氨酸和内源吲哚乙酸的生物合成而实现的。受伤对愈伤组织的形成也起了重要的协同作用。     

L-色氨酸

<正> L-色氨酸是Hopkins和Kole于1901年发现和分离出来的氨基酸。它是一个含吲哚基的芳香族氨基酸。其化学名称为α-氨基—β-吲哚丙酸。在天然蛋白质中,一般含量甚低。含量较高的蛋、奶、肉类等动物蛋白仍不超过1%,植物蛋白中含量一般很低。特别在玉米、木茨、葫萝卜中,色氨酸含量几乎测不出来。此外,还以一些衍生物存在于自然界,如:可可的干酪苦味就是与     

L-色氨酸

<正> L-色氨酸是Hopkins和Kole于1901年发现和分离出来的氨基酸。它是一个含吲哚基的芳香族氨基酸。其化学名称为α-氨基—β-吲哚丙酸。在天然蛋白质中,一般含量甚低。含量较高的蛋、奶、肉类等动物蛋白仍不超过1%,植物蛋白中含量一般很低。特别在玉米、木茨、葫萝卜中,色氨酸含量几乎测不出来。此外,还以一些衍生物存在于自然界,如:可可的干酪苦味就是与     

Cytochrome P450 CYP79B2 from Arabidopsis catalyzes the conversion of tryptophan to indole-3-acetaldoxime, a precursor of indole glucosinolates and indole-3-acetic acid

Glucosinolates are natural plant products known as flavor compounds, cancer-preventing agents, and biopesticides. We report cloning and characterization of the cytochrome P450 CYP79B2 from Arabidopsis. Heterologous expression of CYP79B2 in Escherichia coli shows that CYP79B2 catalyzes the conversion of tryptophan to indole-3-acetaldoxime. Recombinant CYP79B2 has a K(m) of 21 microm and a V(max) of 7.78 nmol/h/ml culture. Inhibitor studies show that CYP79B2 is different from a previously described enzyme activity that converts tryptophan to indole-3-acetaldoxime (Ludwig-Müller, J. , and Hilgenberg, W. (1990) Phytochemistry, 29, 1397-1400). CYP79B2 is wound-inducible and expressed in leaves, stem, flowers, and roots, with the highest expression in roots. Arabidopsis overexpressing CYP79B2 has increased levels of indole glucosinolates, which strongly indicates that CYP79B2 is involved in indole glucosinolate biosynthesis. Our data show that oxime production by CYP79s is not restricted to those amino acids that are precursors for cyanogenic glucosides. Our data are consistent with the hypothesis that indole glucosinolates have evolved from cyanogenesis. Indole-3-acetaldoxime is a precursor of the plant hormone indole-3-acetic acid, which suggests that CYP79B2 might function in biosynthesis of indole-3-acetic acid. Identification of CYP79B2 provides an important tool for modification of the indole glucosinolate content to improve nutritional value and pest resistance.     

拟南芥色氨酸与吲哚乙酸生物合成的研究进展

拟南芥色氨酸生物合成途径的研究已逐渐成为植物分子生物学家了解植物基因结构和表达调控最主要的模式系统之一。到目前为止,编码拟南芥色氨酸合成途径的七种酶蛋白的基因已经全部被克隆,并进行了不同程度的分子生物学研究。长期以来,色氨酸一直被认为是植物生长素吲哚乙酸（ＩＡＡ）生物合成（从头合成）的前体物,但近年来人们发现生长素合成的非色氨酸途径可能是其在植物中生物合成的主要途径。植物在不同的发育阶段可能采用不同的方式合成ＩＡＡ。     

Characterization of TioF, a tryptophan 2,3-dioxygenase involved in 3-hydroxyquinaldic acid formation during thiocoraline biosynthesis

Thiocoraline is a thiodepsipeptide antitumor agent that belongs to the family of bisintercalator natural products that bind duplex DNA through their two planar intercalating moieties. In thiocoraline, the 3-hydroxyquinaldic acid (3HQA) chromophores required for intercalation are derived from L-Trp. We have expressed the Micromonospora sp. ML1 tryptophan 2,3-dioxygenase(TDO) TioF, purified it from E. coli, and confirmed its role in the irreversible oxidation of L-Trp to N-formylkynurenine, the proposed first step during 3HQA biosynthesis. We have established that TioF is a catalyst with broader specificity than other TDOs, but that is less promiscuous than indoleamine 2,3-dioxygenases. TioF was found to display activity with various L-Trp analogs (serotonin, D-Trp, and indole). The TioF reaction products generated during this study will be used as substrates for subsequent analysis of the other enzymes involved in 3HQA biosynthesis.     

Development of indole chemistry to label tryptophan residues in protein for determination of tryptophan surface accessibility

Solvent accessibility can be used to evaluate protein structural models, identify binding sites, and characterize protein conformational changes. The differential modification of amino acids at specific sites enables the accessible surface residues to be identified by mass spectrometry. Tryptophan residues within proteins can be differentially labeled with halocompounds by a photochemical reaction. In this study, tryptophan residues of carbonic anhydrase are reacted with chloroform, 2,2,2-trichloroethanol (TCE), 2,2,2-trichloroacetate (TCA), or 3-bromo-1-propanol (BP) under UV irradiation at 280 nm. The light-driven reactions with chloroform, TCE, TCA, and BP attach a formyl, hydroxyethanone, carboxylic acid, and propanol group, respectively, onto the indole ring of tryptophan. Trypsin and chymotrypsin digests of the modified carbonic anhydrase are used to map accessible tryptophan residues using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Tryptophan reactivity is determined by identifying peptides with tryptophan residues modified with the appropriate label. The reactivity is calculated from the frequency that the modification is identified and a semiquantitative measure of the amount of products formed. Both of these measures of tryptophan reactivity correlate significantly with the accessible surface area of tryptophan residues in carbonic anhydrase determined from the X-ray crystal structure. Therefore the photochemical reaction of halocompounds with tryptophan residues in carbonic anhydrase indicates the degree of solvent accessibility of these residues.     

Influence of steric bulk and electrostatics on the hydroxylation regiospecificity of tryptophan hydroxylase: characterization of methyltryptophans and azatryptophans as substrates

Tryptophan hydroxylase is a pterin-dependent amino acid hydroxylase that catalyzes the incorporation of one atom of molecular oxygen into tryptophan to form 5-hydroxytryptophan. The substrate specificity and hydroxylation regiospecificity of tryptophan hydroxylase have been investigated using tryptophan analogues that have methyl substituents or nitrogens incorporated into the indole ring. The products of the reactions show that the regiospecificity of tryptophan hydroxylase is stringent. Hydroxylation does not occur at the 4 or 6 carbon in response to changes in substrate topology or atomic charge. 5-Hydroxymethyltryptophan and 5-hydroxy-4-methyltryptophan are the products from 5-methyltryptophan. These products establish that the hydroxylating intermediate is sufficiently potent to hydroxylate benzylic carbons and that the direction of the NIH shift in tryptophan hydroxylase is from carbon 5 to carbon 4. The effects on the V/K values for the amino acids indicate that the enzyme is most sensitive to changes at position 5 of the indole ring. The V(max) values for amino acid hydroxylation differ at most by a factor of 3 from that observed for tryptophan, while the efficiencies of hydroxylation with respect to tetrahydropterin consumption vary 6-fold, consistent with oxygen transfer to the amino acid being partially or fully rate limiting in productive catalysis.     

Metabolic engineering in Nicotiana benthamiana reveals key enzyme functions in Arabidopsis indole glucosinolate modification

Indole glucosinolates, derived from the amino acid Trp, are plant secondary metabolites that mediate numerous biological interactions between cruciferous plants and their natural enemies, such as herbivorous insects, pathogens, and other pests. While the genes and enzymes involved in the Arabidopsis thaliana core biosynthetic pathway, leading to indol-3-yl-methyl glucosinolate (I3M), have been identified and characterized, the genes and gene products responsible for modification reactions of the indole ring are largely unknown. Here, we combine the analysis of Arabidopsis mutant lines with a bioengineering approach to clarify which genes are involved in the remaining biosynthetic steps in indole glucosinolate modification. We engineered the indole glucosinolate biosynthesis pathway into Nicotiana benthamiana, showing that it is possible to produce indole glucosinolates in a noncruciferous plant. Building upon this setup, we demonstrate that all members of a small gene subfamily of cytochrome P450 monooxygenases, CYP81Fs, are capable of carrying out hydroxylation reactions of the glucosinolate indole ring, leading from I3M to 4-hydroxy-indol-3-yl-methyl and/or 1-hydroxy-indol-3-yl-methyl glucosinolate intermediates, and that these hydroxy intermediates are converted to 4-methoxy-indol-3-yl-methyl and 1-methoxy-indol-3-yl-methyl glucosinolates by either of two family 2 O-methyltransferases, termed indole glucosinolate methyltransferase 1 (IGMT1) and IGMT2.