植物丝氨酸羧肽酶及其类蛋白的研究进展

丝氨酸羧肽酶（serine carboxypeptidases,SCP）是一类属于α/β水解酶家族的蛋白酶,在植物生长发育过程中参与多肽和蛋白质的加工、修饰与降解等多个重要环节,并且在许多生化途径包括次生代谢产物的生物合成、催化酰基转移、除草剂共轭和种子萌发相关蛋白质降解中起重要作用。介绍和评述了植物丝氨酸羧肽酶及其类蛋白的种类、特点、功能及其基因的表达调控,并对植物丝氨酸羧肽酶及其类蛋白的研究方向和应用提出了展望。     

羟基肉桂酰基转移酶的结构、功能与应用

羟基肉桂酰基转移酶（hydroxycinnamoyl transferase,HCT）属于植物酰基转移酶家族的一个重要分支,具有“HXXXD”和“DFGWG”两个保守序列,以多种酰基辅酶A（肉桂酰辅酶A、对香豆酰辅酶A、咖啡酰辅酶A、阿魏酰辅酶A和芥子酰辅酶A等）作为酰基供体,催化多种底物（莽草酸、奎尼酸、4 羟基苯乳酸、龙胆酸和4 羟基苯乙胺等）形成酯类或酰胺化合物。其酰基化产物可改善植物次生代谢产物的理化性质和生物活性,因此HCT被广泛应用于开发生物质能源、改良作物品种和研制抗炎药物,对植物次生代谢产物的合成与后修饰具有重要意义。本文系统介绍了HCT的序列特点、蛋白质结构特征、酰基化反应机制和其在工业、农业及医药行业的应用,并对HCT的未来发展前景进行了展望。     

植物二酰甘油酰基转移酶基因(DGAT)研究进展

三酰甘油(TAG)是油料作物最主要的储藏脂类,二酰甘油酰基转移酶(DGAT,EC2.3.1.20)是TAG合成途径的限速酶,其主要作用是催化二酰甘油加上酰基脂肪酸形成三酰甘油.在植物中已发现了3种不同类型的DGAT基因,分别为DGAT1、DGAT2和DGAT3.该文对近年来国内外有关植物DGAT相关基因及其蛋白分类、定位、结构及其在脂肪酸合成、种子发育与萌发、幼苗发育、叶片新陈代谢等过程中的作用等研究进展进行综述.为提高油料作物种子油含量以及特定脂肪酸积累提供理论参考.     

生物法合成辛伐他汀

通过提高E.coli BL21(DE3)/pAW31菌株中的酰基转移酶LovD的表达,并以Monacolin J为底物,催化合成辛伐他汀。考察发酵培养基和发酵条件对酰基转移酶LovD表达的影响;采用SDS-PAGE凝胶电泳法检测酰基转移酶LovD表达情况;并建立酶活测定方法,测定酰基转移酶LovD的实际酶活。通过实验确定酰基转移酶LovD摇瓶发酵的最佳条件:发酵培养基为TB培养基,接种量为4%,诱导初始菌密度为0.7(OD600)I,PTG浓度为0.2 mmol/L,在20℃下诱导20 h。在最佳条件下,酰基转移酶LovD的表达水平为100 mg/L,辛伐他汀的产量为1.2 g/L。     

膜脂组成与植物抗冷性的关系及其分子生物学研究进展

植物的抗冷性与膜脂的组分和结构密切相关,与质膜中脂肪酸的不饱和度关系更为密切。膜脂不饱和脂肪酸含量越高,膜脂相变温度越低,植物的抗冷性提高。植物体内存在一些降低膜脂脂肪酸饱和程度的酶,如甘油3磷酸酰基转移酶,ω3脂肪酸去饱和酶等,它们能够催化膜中脂肪酸的去饱和反应,生成不饱和脂肪酸,从而提高植物的抗冷性。本文就低温对膜脂的影响、膜脂组成与植物抗寒性的关系及其分子生物学研究进展作一简单综述。     

酰基转移酶研究

酰基转移酶(acyltransferase)是一个多功能蛋白质大家族,在机体内组蛋白酰基转移酶、N-乙酰转移酶、胆固醇酰基转移酶等对维持机体正常功能与疾病发生都密切相关,研究其功能与机制对于疾病的发病机理与临床治疗具有重要意义.     

植物萜类生物合成中的后修饰酶

萜类化合物由于其结构类型丰富多样而被称为"terpenome".除了参与植物生长发育、环境应答等生理过程,萜类化合物还应用于医药、有机化工等领域.萜类的生物合成大致可分为前体形成、骨架构建以及后修饰三部分,基本骨架通常由萜类合酶催化形成,进一步在后修饰酶的作用下产生数以万计的萜类化合物.结合我们对香茶菜二萜生物合成的初步研究结果,本文主要针对近年来植物萜类生物合成中的一些有代表性的后修饰酶包括P450单氧酶、双键还原酶、酰基转移酶和糖基转移酶,进行研究现状分析与展望.     

螺旋霉素3-O-酰基转移酶基因的删除和主要产生螺旋霉素组分Ⅰ菌株的获得

螺旋霉素(SP)为16元环大环内酯类抗生素,含有螺旋霉素Ⅰ、Ⅱ和Ⅲ个组分,其结构的差异为16元内酯环的C3上分别连接羟基(SPⅠ)、乙酰基(SPⅡ)和丙酰基(SPⅢ);SPⅡ和SPⅢ是在相同的3-O-酰基转移酶催化下SPⅠ进一步酰化的产物。SPⅠ、SPⅡ和SPⅢ在生物学活性方面无大差异。为简化螺旋霉素组分,便于今后对其结构进行进一步改造,根据碳霉素和麦迪霉素生物合成中的3-O-酰基转移酶序列,设计了简并性PCR引物,并采用SON-PCR(single oligonucleotide nested PCR)方法,从螺旋霉素产生菌S.spiramyceticus F21中进行特异性扩增,获得了螺旋霉素3-O-酰基转移酶基因(sspA)及其侧翼序列,共约4.3kb(其中的3457nt DNA序列已被Genbank收录,DQ642742)。采用DNA同源双交换技术对S.spiramyceticus F21中的sspA进行了删除。对螺旋霉素原株和sspA缺失变株进行发酵产物提取和HPLC分析表明:原株中SPⅠ、SPⅡ和SPⅢ的相对含量分别为7.8%、67%和25%,变株中则分别为72%、18%和9.6%;变株主要组分为SPⅠ。螺旋霉素sspA缺失变株的获得为螺旋霉素组分简化及其衍生物的结构改造奠定了基础。     

植物sirtuin蛋白家族研究进展

植物sirtuin蛋白家族是一类依赖β-烟酰胺腺嘌呤二核苷酸(β-NAD⁺)、分别催化非组蛋白和组蛋白赖氨酸上Nε-乙酰基和Nε-酰基(乙酰基、巴豆酰基、丁酰基和2-羟基异丁酰基)去除的酶,具有调控非组蛋白活性和基因表达的功能。已证明sirtuin蛋白家族成员在水稻(Oryza sativa)、大豆(Glycine max)和番茄(Solanum lycopersicum)等植物的生长发育以及盐、冷、热和病原菌等胁迫响应中发挥重要的表观调控作用。该文对植物sirtuins的酶活性、催化底物、细胞定位和功能进行综述,为理解其表观遗传调控机制及丰富新功能研究提供参考。     

酰基供体结构对褶皱念珠茵脂肪酶（CRL）催化芳香仲醇动力学拆分立体选择性的影响

研究仲醇的酶催化动力学拆分机制,发现酰基供体的结构是影响酶催化动力学拆分选择性的一个重要因素。通过实验发现了一类用于仲醇动力学拆分（KR）的优秀酰基供体——长链有机酸的对氯苯酚酯,并将这种酰基供体成功用于褶皱念珠菌脂肪酶（CRL）催化的仲醇动力学拆分过程。在1-苯乙醇的动力学拆分（KR）过程中,随着对氯苯酚有机酸酯供体中酰基部分碳原子数的增加,产物的对映体过量值（e．e.p值）也在不断地提高。当碳原子数≥5,转化率达到50％时,产物的叫．。值仍能保持大于99％。这样的规律也适用于其他的仲醇拆分过程,当选择对氯苯酚戊酸酯作为酰基供体用于其他仲醇的动力学拆分过程时,可以实现仲醇的高效拆分,反应6h转化率达到50％,产物的对映体过量值e．e．p为100％。     

Application of LC/MS systems to structural characterization of flavonoid glycoconjugates

Mass spectrometry is currently one of the most versatile and sensitive instrumental methods applied to structural characterization of plant secondary metabolite mixtures isolated from biological material. Plant tissues contain thousands of natural products fulfilling different roles in plant physiology and biochemistry. These natural products have various biological activities in respect to plants synthesizing them, in their responses to different environmental stresses and are also active principles of food supplements and pharmaceuticals of plant origin. Flavonoids constitute a large group of phenolic secondary metabolites and are probably produced by all terrestrial plant species. More than 9000 glycoconjugates of flavonoids are presently known in the plant kingdom and more than 50 of them may be present in a single plant. For this reason methods of identification and analysis of this group of compounds are particularly demanded. Due to a high number of metabolites present in plant extracts, the isolation and purification of most compounds in amounts suitable for unambiguous characterization with NMR methods is often impossible. For these reasons elaboration of strategies for sufficiently precise structural characterization of compounds present in mixture samples is currently a primary task. Mass spectrometry, thanks to application of different physical phenomena for ionization, separation and detection of analyzed molecules, became the method of choice among analytical methods applied for identification, structural characterization and quantitative analysis of the natural products. Methods of analysis of differently substituted flavonoids (O- and C-glycosides, differentiation of various oligosaccharidic substituents, detection of acylated compounds) are presented in the paper. A proper application of mass spectrometric methods in well-defined and strictly controlled technical parameters of analysis permits obtaining important structural information. Among others, recording collision induced dissociation mass spectra allows identification of compounds after comparison of the registered MS spectra with these present in the existing databases.     

Glucose ester enabled acylation in plant specialized metabolism

Acylation of core compound skeletons, together with other modifications, plays a significant role in producing the incredible diversity of plant specialized metabolites. Two major classes of acyltransferases, the BAHD and serine carboxypeptidase-like (SCPL) acyltransferases, can bring together through acylation compounds from the same or divergent metabolic pathways. BAHD acyltransferases (BAHD-ATs) employ CoA thioesters as the activated substrate, SCPL acyltransferases (SCPL-ATs), on the other hand, utilize β-acetal esters, typically glucose esters formed by UDP glycosyltransferases (UGTs). While the general trend of high energy glucose ester enabled acyltransfers is seen throughout the spermatophytes (seed plants), the specific metabolites that are conjugated appear to be lineage specific. In this review, we examine the reaction mechanism, biochemical property and evolutionary relationship of SCPL-ATs that utilize various glucose ester donors and acceptors from the same or different plant specialized metabolic pathways. The occurrence and taxonomic distribution of galloylated flavan-3-ols, hydrolyzable tannins and galloylated flavonols are also evaluated. Furthermore, glucose ester (acyl donor)-forming UGT activities and the subcellular localization of the UGT and SCPL-AT catalyzed reactions are discussed.     

Acyltransferases in plants: a good time to be BAHD

Acylation is a common and biochemically significant modification of plant secondary metabolites. Plant BAHD acyltransferases constitute a large family of acyl CoA-utilizing enzymes whose products include small volatile esters, modified anthocyanins, as well as constitutive defense compounds and phytoalexins. The catalytic versatility of BAHD enzymes makes it very difficult to make functional predictions from primary sequence alone. Recent advances in genome sequencing and the availability of the first crystal structure of a BAHD member are, however, providing insights into the evolution and function of these acyltransferases within the plant kingdom.     

Molecular approaches for improvement of medicinal and aromatic plants

Medicinal and aromatic plants (MAPs) are important sources for plant secondary metabolites, which are important for human healthcare. Improvement of the yield and quality of these natural plant products through conventional breeding is still a challenge. However, recent advances in plant genomics research has generated knowledge leading to a better understanding of the complex genetics and biochemistry involved in biosynthesis of these plant secondary metabolites. This genomics research also concerned identification and isolation of genes involved in different steps of a number of metabolic pathways. Progress has also been made in the development of functional genomics resources (EST databases and micro-arrays) in several medicinal plant species, which offer new opportunities for improvement of genotypes using perfect markers or genetic transformation. This review article presents an overview of the recent developments and future possibilities in genetics and genomics of MAP species including use of transgenic approach for their improvement.     

Characterization of PPTNs, a cyanobacterial phosphopantetheinyl transferase from Nodularia spumigena NSOR10

The phosphopantetheinyl transferases (PPTs) are a superfamily of essential enzymes required for the synthesis of a wide range of compounds, including fatty acids, polyketides, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways by transfer of a phosphopantetheinyl moiety. The diverse PPT superfamily can be divided into two families based on specificity and conserved sequence motifs. The first family is typified by the Escherichia coli acyl carrier protein synthase (AcpS), which is involved in fatty acid synthesis. The prototype of the second family is the broad-substrate-range PPT Sfp, which is required for surfactin biosynthesis in Bacillus subtilis. Most cyanobacteria do not encode an AcpS-like PPT, and furthermore, some of their Sfp-like PPTs belong to a unique phylogenetic subgroup defined by the PPTs involved in heterocyst differentiation. Here, we describe the first functional characterization of a cyanobacterial PPT based on a structural analysis and subsequent functional analysis of the Nodularia spumigena NSOR10 PPT. Southern hybridizations suggested that this enzyme may be the only PPT encoded in the N. spumigena NSOR10 genome. Expression and enzyme characterization showed that this PPT was capable of modifying carrier proteins resulting from both heterocyst glycoplipid synthesis and nodularin toxin synthesis. Cyanobacteria are a unique and vast source of bioactive metabolites; therefore, an understanding of cyanobacterial PPTs is important in order to harness the biotechnological potential of cyanobacterial natural products.     

Serine carboxypeptidase-like acyltransferases

In plant secondary metabolism, an alternative pathway of ester formation is facilitated by acyltransferases accepting 1-O-beta-acetal esters (1-O-beta-glucose esters) as acyl donors instead of coenzyme A thioesters. Molecular data indicate homology of these transferases with hydrolases of the serine carboxypeptidase type defining them as serine carboxypeptidase-like (SCPL) acyltransferases. During evolution, they apparently have been recruited from serine carboxypeptidases and adapted to take over acyl transfer function. SCPL acyltransferases belong to the highly divergent class of alpha/beta hydrolases. These enzymes make use of a catalytic triad formed by a nucleophile, an acid and histidine acting as a charge relay system for the nucleophilic attack on amide or ester bonds. In analogy to SCPL acyltransferases, bacterial thioesterase domains are known which favour transferase activity over hydrolysis. Structure elucidation reveals water exclusion and a distortion of the oxyanion hole responsible for the changed activity. In plants, SCPL proteins form a large family. By sequence comparison, a distinguished number of Arabidopsis SCPL proteins cluster with proven SCPL acyltransferases. This indicates the occurrence of a large number of SCPL proteins co-opted to catalyse acyltransfer reactions. SCPL acyltransferases are ideal systems to investigate principles of functional adaptation and molecular evolution of plant genes.     

植物萜类次生代谢及其调控

植物次生代谢在植物生长发育、环境适应、抵御病虫害等方面发挥着重要作用,这些天然产物组成地球上最丰富的有机化合物的宝库．萜类是植物代谢产物中种类最多的一类,具有重要的生理和生态功能,一些成分还有应用价值．近十几年来,人们在萜类化合物的分离、鉴定、应用、生物合成、相关基因与基因族、酶蛋白结构和功能、代谢调控以及代谢工程等各方面取得了重大进展．本文概述了植物萜类化合物代谢及其调控领域的研究进展与发展趋势．     

Structural insights into biosynthesis of resorcinolic lipids by a type III polyketide synthase in Neurospora crassa

Microbial type III polyketide synthases (PKSs) have revealed remarkable mechanistic as well as functional versatility. Recently, a type III PKS homolog from Azotobacter has been implicated in the biosynthesis of resorcinolic lipids, thus adding a new functional significance to this class of proteins. Here, we report the structural and mutational investigations of a novel type III PKS protein from Neurospora crassa involved in the biosynthesis of resorcinolic metabolites by utilizing long chain fatty acyl-CoAs. The structure revealed a long hydrophobic tunnel responsible for its fatty acyl chain length specificity resembling that of PKS18, a mycobacterial type III PKS. Structure-based mutational studies to block the tunnel not only altered the fatty acyl chain specificity but also resulted in change of cyclization pattern affecting the product profile. This first structural characterization of a resorcinolic lipid synthase provides insights into the coordinated functioning of cyclization and a substrate-binding pocket, which shows mechanistic intricacy underlying type III PKS catalysis.     

植物次生代谢及其与环境的关系

人类对植物次生代谢产物（天然产物）的早期研究源于它们的应用价值,近些年来人们越来越认识到植物次生代谢产物广泛的生物学效应,开始重新评价这些化合物在植物生命活动以及生态系统中可能扮演的角色。植物的次生代谢是植物在长期进化中与环境（生物的和非生物的）相互作用的结果,次生代谢产物在植物提高自身保护和生存竞争能力、协调与环境关系上充当着重要的角色。介绍了植物次生代谢及其产物的特点,概述了植物次生代谢与温度、水分、光照、养分、CO2浓度、UV-B辐射、环境污染等非生物环境以及与化学防御、化感作用、菌根共生、微生物病害的关系。研究植物次生代谢与环境的关系,可以从更深的层次发掘植物与环境的内在联系,为全面、深入认识植物与环境的相互关系提供新的研究途径,同时也有利于人类更有效、合理地利用植物的次生代谢产物。