芳香族异戊烯转移酶的研究进展

异戊烯基转移酶（prenyltransferase）催化异戊烯基转移至异戊烯单元、芳香环或蛋白质上。芳香族异戊烯基转移酶将异戊烯单元融入含有芳环的化合物,从而形成具有重要生物学功能的各类活性分子,如泛醌、质体醌、维生素E、异戊烯黄酮类以及真菌代谢物等。该文综述了近年来植物和真菌芳香族异戊烯转移酶的分子生物学研究进展,包括膜结合的参与质体醌生物合成的homogentisate solanesyltransferase、参与维生素E生物合成的homogentisate phytyltransferase、类黄酮异戊烯转移酶（flavonoid prenyltransferase）和可溶性的真菌吲哚异戊烯转移酶等。     

植物中异戊烯基取代的酚类化合物及其分布

异戊烯基取代的酚类化合物由UbiA家族的异戊烯基转移酶(prenyltransferase)催化异戊烯基转移到芳香族化合物母核上,其亲脂性较无异戊烯基取代的化合物明显增强,并提高了与生物膜的亲和力,从而形成了各种具有重要生物学功能的活性分子,在植物防御和人体健康方面具有重要作用。本综述总结了538种异戊烯基酚类化合物的取代基类别和取代位点等,为发掘植物中新型异戊烯基转移酶,以及受体和供体的选择提供参考,以期有更多异戊烯基转移酶可应用于合成生物学来生产具有重要活性的异戊烯基酚类化合物。该文对国内外报道的378种类黄酮,80种香豆素类,27种醌类,32种二苯乙烯类,16种对羟基苯甲酸类,5种苯丙酸类共计538种异戊烯基取代的酚类化合物的取代基类别、取代位置以及在植物中的分布进行总结,发现异戊烯基酚类化合物主要分布在28个科中,且以C5和C10取代基为主。该文还综述了已鉴定功能的30余种植物芳香族异戊烯基转移酶。     

粗毛淫羊藿的类黄酮异戊烯转移酶基因的克隆与表达分析

药用植物淫羊藿富含异戊烯类黄酮,LC-MS定量分析表明,3～4月份粗毛淫羊藿叶片处于变色期时淫羊藿苷含量最高。通过简并引物和RACE-PCRAL粗毛淫羊藿叶片获得异戊烯转移酶同源基因（命名为EaPTI）。序列分析表明,EaPTI与维生素E合成相关的尿黑酸异戊烯转移酶位于同一进化枝,靠近类黄酮异戊烯转移酶。RT-PCR结果显示,EaPTI在叶片和幼茎中表达显著。     

桑叶中聚戊烯醇结构与含量测定

本文建立了高效液相色谱法确定桑叶聚戊烯醇的异戊烯基单元数和含量的方法,采用Thermo C18ODS-2(250 mm×4.6 mm,5μm)色谱柱,流动相为甲醇-异丙醇(8∶7,v/v),流速为1.50 mL/min,柱温为25℃,检测器为PDA。测定结果表明,桑叶中聚戊烯醇的异戊烯基单元数为10~12,聚戊烯醇含量为0.731%。该方法准确度高、重现性好,能快速测定桑叶中聚戊烯醇的异戊烯基单元数和含量,为桑叶资源的开发提供理论依据。     

植物中的异戊烯基转移酶

介绍了近年来植物中异戊烯基转移酶(又称异戊烯基二磷酸合酶,IPPS)的研究进展,着重讨论IPPS的链长决定机制,同时分析了有待解决的问题.     

丹参中尿黑酸茄尼酯转移酶基因的克隆及其生物信息学分析

尿黑酸茄尼酯转移酶(homogentisate solanesyltransferase, HST)是一种尿黑酸异戊烯基转移酶(HPT),催化尿黑酸和茄尼醇焦磷酸的聚合反应,为植物中质体醌9 (PQ_9)合成的关键酶。本实验利用RACE技术克隆了丹参中HST基因cDNA,将其命名为SmHST。SmHST的开放阅读框(ORF)为1 137 bp,编码378个氨基酸残基,蛋白大小为41.75 kD,等电点(PI)为9.68。SmHST为膜结合蛋白,具有复杂的高级结构。亚细胞定位预测该蛋白定位于叶绿体中,这和PQ_9合成部位是一致的。Real-time PCR分析SmHST基因在丹参嫩叶中表达量最高。SmHST响应PEG6000和Me JA处理,说明SmHST可能参与了丹参的抗逆过程。     

银杏叶聚戊烯醇的化学、纯化和药效研究

通过石油醚提取、水解、萃取和柱层析等分离,制得银杏叶叶聚戊烯醇纯样,其化学结构由NMR、IR和MS鉴定为桦木聚戊烯醇,异戊烯基单元数为15－21。另外,对聚戊烯醇的纯化工艺和毒、药理等进行了研究,提出纯化70％以上聚戊烯醇的方法,适合大指生产,为聚戊烯醇制剂的开发提供基础。     

蛋白质异戊烯化修饰与抗肿瘤研究

蛋白质经过异戊烯化翻译后修饰正确的定位于膜上,在生物体内的信号转导过程（如异三聚体G蛋白）中起非常重要作用。其修饰过程包括异戊烯化、蛋白水解、甲基化以及棕榈化等。目前已知主要有3种蛋白质异戊烯化转移酶：法尼酰基转移酶（FTase）、二牛龙牛儿基转移酶I（GGTaseI）、二牛龙牛儿基转移酶Ⅱ（GGTaseII）。作用于异戊烯化修饰的关键酶或下游因素是目前抗肿瘤的主要策略之一。     

微生物异戊烯基转移酶研究进展

类异戊二烯(isoprenoids)是最具化学多样性的一种天然分子家族，参与微生物中类胡萝卜素、甾醇等次生代谢物的合成，这类物质在工业大规模生产中具有广阔的商业前景。异戊烯基转移酶是类异戊二烯合成途径中的关键酶，其活性及编码基因的转录水平参与调节次生代谢物产量，在类异戊二烯化合物生物合成途径中发挥重要作用。本文重点归纳了微生物中异戊烯基转移酶的发现与鉴定，分析其结构特点与链长决定机制，讨论异戊烯基转移酶家族之间的复杂进化，概述酶基因表达调控的应用以及生物合成研究现状，为深入研究异戊烯基转移酶作用机理及各领域中的应用提供思路。     

落叶松和水杉针叶的聚戊烯醇

从落叶松（Ｌａｒｉｘｇｍｅｌｉｎｉ（Ｒｕｐｒ．）Ｒｕｐｒ．）和水杉（ＭｅｔａｓｅｑｕｏｉａｇｌｙｐｔｏｓｔｒｏｂｏｉｄｅｓＨｕｅｔＣｈｅｎｇ）针叶中首次分离出聚戊烯乙酸酯,其结构由１ＨＮＭＲ和１３ＣＮＭＲ鉴定为桦木聚戊烯醇型（ｂｅｔｕｌａｐｒｅｎｏｌ）,其异戊烯基结构单元数（ｎ）与ＨＰＬＣ的ｌｏｇｔｒ线性关系研究确定ｎ在１０～２４间     

Prenylation of aromatic compounds,a key diversification of plant secondary metabolites

Prenylation plays a major role in the diversification of aromatic natural products, such as phenylpropanoids, flavonoids, and coumarins. This biosynthetic reaction represents the crucial coupling process of the shikimate or polyketide pathway providing an aromatic moiety and the isoprenoid pathway derived from the mevalonate or methyl erythritol phosphate (MEP) pathway, which provides the prenyl (isoprenoid) chain. In particular, prenylation contributes strongly to the diversification of flavonoids, due to differences in the prenylation position on the aromatic rings, various lengths of prenyl chain, and further modifications of the prenyl moiety, e.g., cyclization and hydroxylation, resulting in the occurrence of ca. 1000 prenylated flavonoids in plants. Many prenylated flavonoids have been identified as active components in medicinal plants with biological activities, such as anti-cancer, anti-androgen, anti-leishmania, and anti-nitric oxide production. Due to their beneficial effects on human health, prenylated flavonoids are of particular interest as lead compounds for producing drugs and functional foods. However, the gene coding for prenyltransferases that catalyze the key step of flavonoid prenylation have remained unidentified for more than three decades, because of the membrane-bound nature of these enzymes. Recently, we have succeeded in identifying the first prenyltransferase gene SfN8DT-1 from Sophora flavescens, which is responsible for the prenylation of the flavonoid naringenin at the 8-position, and is specific for flavanones and dimethylallyl diphosphate (DMAPP) as substrates. Phylogenetic analysis showed that SfN8DT-1 has the same evolutionary origin as prenyltransferases for vitamin E and plastoquinone. A prenyltransferase GmG4DT from soybean, which is involved in the formation of glyceollin, was also identified recently. This enzyme was specific for pterocarpan as its aromatic substrate, and (?)-glycinol was the native substrate yielding the direct precursor of glyceollin I. These enzymes are localized to plastids and the prenyl chain is derived from the MEP pathway. Further relevant genes involved in the prenylation of other types of polyphenol are expected to be cloned by utilizing the sequence information provided by the above studies.     

Evolution of aromatic prenyltransferases in the biosynthesis of indole derivatives

A series of putative indole prenyltransferase genes could be identified in the genome sequences of different fungal strains including Aspergillus fumigatus and Neosartorya fischeri. The gene products show significant sequence similarities to dimethylallyltryptophan synthases from different fungi. We have cloned and overexpressed seven of these genes, fgaPT1, fgaPT2, ftmPT1, ftmPT2, 7-dmats, cdpNPT and anaPT in Escherichia coli and Saccharomyces cerevisiae. The overproduced enzymes were characterised biochemically. Three additional indole prenyltransferases, DmaW-Cs, TdiB and MaPT were also identified and characterised in the last years. Sequence analysis and comparison with known aromatic prenyltransferases as well as biochemical investigation revealed that these enzymes belong to a group of aromatic prenyltransferases. The characterised prenyltransferases are soluble proteins, catalyse different prenyl transfer reactions on indole moieties of various substrates and do not require divalent metal ions for their prenyl transfer reactions. In addition, indole prenyltransferases carry tryptophan aminopeptidase activity, which strengths their relationship in the evolution. These properties differ clearly from membrane-bound aromatic prenyltransferases from different sources and soluble prenyltransferases from bacteria. All of the indole prenyltransferases accepted only dimethylallyl diphosphate as prenyl donor. On the other hand, they showed broad substrate specificity towards their aromatic substrates. Diverse simple tryptophan derivatives and tryptophan-containing cyclic dipeptides were accepted by these enzymes, providing a strategy for convenient production of biologically active substances, e.g. by chemoenzymatic synthesis.     

Prenyltransferases as key enzymes in primary and secondary metabolism

Attachment of isoprene units to various acceptors by prenylation plays an important role in primary and secondary metabolism of living organisms. Protein prenylation belongs to posttranslational modification and is involved in cellular regulation process. Prenylated secondary metabolites usually demonstrate promising biological and pharmacological activities. Prenyl transfer reactions catalyzed by prenyltransferases represent the key steps in the biosynthesis and contribute significantly to the structural and biological diversity of these compounds. In the last decade, remarkable progress has been achieved in the biochemical, molecular, and structural biological investigations of prenyltransferases, especially on those of the members of the dimethylallyltryptophan synthase (DMATS) superfamily. Until now, more than 40 of such soluble enzymes are identified and characterized biochemically. They catalyze usually regioselective and stereoselective prenylations of a series of aromatic substances including tryptophan, tryptophan-containing peptides, and other indole derivatives as well as tyrosine or even nitrogen-free substrates. Crystal structures of a number of prenyltransferases have been solved in the past 10 years and provide a solid basis for understanding the mechanism of prenyl transfer reactions.     

Prenyl transfer to aromatic substrates in the biosynthesis of aminocoumarins,meroterpenoids and phenazines: The ABBA prenyltransferase family

Aromatic prenyltransferases transfer prenyl moieties onto aromatic acceptor molecules, catalyzing an electrophilic substitution of the aromatic ring under formation of carbon–carbon bonds. They give rise to an astounding diversity of primary and secondary metabolites in plants, fungi and bacteria. This review describes a recently discovered family of aromatic prenyltransferases. The structure of these enyzmes shows a type of β/α fold with antiparallel β strands. Due to the α-β-β-α architecture of this fold, this group of enzymes was designated as ABBA prenyltransferases. They lack the (N/D)DxxD motif which is characteristic for many other prenyltransferases.At present, 14 genes with sequence similarity to ABBA prenyltransferases can be identified in the database. A phylogenetic analysis of these genes separates them into two clades. One of them comprises the 4-hydroxyphenylpyruvate 3-dimethylallyltransferases CloQ and NovQ involved in aminocoumarin antibiotic biosynthesis in Streptomyces strains, as well as four genes of unknown function from fungal genomes. The other clade comprises genes involved in the biosynthesis of prenylated naphthoquinones and prenylated phenazines in different streptomycetes. ABBA prenyltransferases are soluble biocatalysts which can easily be obtained as homogeneous proteins in significant amounts. Their substrates are accommodated in a surprisingly spacious central cavity which explains their promiscuity for different aromatic substrates. Therefore, the enzymes of this family represent attractive tools for the chemoenzymatic synthesis of bioactive molecules.     

Molecular and structural basis of metabolic diversity mediated by prenyldiphosphate converting enzymes

General thermodynamic calculations using the semiempiric PM3 method have led to the conclusion that prenyldiphosphate converting enzymes require at least one divalent metal cation for the activation and cleavage of the diphosphate–prenyl ester bond, or they must provide structural elements for the efficient stabilization of the intermediate prenyl cation. The most important common structural features, which guide the product specificity in both terpene synthases and aromatic prenyl transferases are aromatic amino acid side chains, which stabilize prenyl cations by cation–π interactions. In the case of aromatic prenyl transferases, a proton abstraction from the phenolic hydroxyl group of the second substrate will enhance the electron density in the phenolic ortho-position at which initial prenylation of the aromatic compound usually occurs.A model of the structure of the integral transmembrane-bound aromatic prenyl transferase UbiA was developed, which currently represents the first structural insight into this group of prenylating enzymes with a fold different from most other aromatic prenyl transferases. Based on this model, the structure–activity relationships and mechanistic aspects of related proteins, for example those of Lithospermum erythrorhizon or the enzyme AuaA from Stigmatella aurantiaca involved in the aurachin biosynthesis, were elucidated. The high similarity of this group of aromatic prenyltransferases to 5-epi-aristolochene synthase is an indication of an evolutionary relationship with terpene synthases (cyclases). This is further supported by the conserved DxxxD motif found in both protein families. In contrast, there is no such relationship to the aromatic prenyl transferases with an ABBA-fold, such as NphB, or to any other known family of prenyl converting enzymes. Therefore, it is possible that these two groups might have different evolutionary ancestors.     

Applications of dimethylallyltryptophan synthases and other indole prenyltransferases for structural modification of natural products

A series of putative indole prenyltransferase genes could be identified in the genome sequences of different fungal strains including Aspergillus fumigatus and Neosartorya fischeri. The gene products show significant sequence similarities to dimethylallyltryptophan synthases from various fungi. These genes belong to different gene clusters and are involved in the biosynthesis of secondary metabolites. Ten of them were cloned and overexpressed in Escherichia coli and Saccharomyces cerevisiae and proven to be soluble proteins. They catalyse different prenyl transfer reactions onto indole moieties of various substrates and do not require divalent metal ions for their prenyl transfer reactions. These enzymes showed broad substrate specificities towards their aromatic substrates. Diverse simple tryptophan derivatives and tryptophan-containing cyclic dipeptides were accepted by several prenyltransferases as substrates and converted to prenylated derivatives. This feature of substrate flexibility was successfully used for regiospecific and stereospecific synthesis of different indole derivatives.     

Cloning and characterization of naringenin 8-prenyltransferase, a flavonoid-specific prenyltransferase of Sophora flavescens

Prenylated flavonoids are natural compounds that often represent the active components in various medicinal plants and exhibit beneficial effects on human health. Prenylated flavonoids are hybrid products composed of a flavonoid core mainly attached to either 5-carbon (dimethylallyl) or 10-carbon (geranyl) prenyl groups derived from isoprenoid (terpenoid) metabolism, and the prenyl groups are crucial for their biological activity. Prenylation reactions in vivo are crucial coupling processes of two major metabolic pathways, the shikimate-acetate and isoprenoid pathways, in which these reactions are also known as a rate-limiting step. However, none of the genes responsible for the prenylation of flavonoids has been identified despite more than 30 years of research in this field. We have isolated a prenyltransferase gene from Sophora flavescens, SfN8DT-1, responsible for the prenylation of the flavonoid naringenin at the 8-position, which is specific for flavanones and dimethylallyl diphosphate as substrates. Phylogenetic analysis shows that SfN8DT-1 has the same evolutionary origin as prenyltransferases for vitamin E and plastoquinone. The gene expression of SfN8DT-1 is strictly limited to the root bark where prenylated flavonoids are solely accumulated in planta. The ectopic expression of SfN8DT-1 in Arabidopsis thaliana resulted in the formation of prenylated apigenin, quercetin, and kaempferol, as well as 8-prenylnaringenin. SfN8DT-1 represents the first flavonoid-specific prenyltransferase identified in plants and paves the way for the identification and characterization of further genes responsible for the production of this large and important class of secondary metabolites.     

Protein prenyltransferases

Three different protein prenyltransferases (farnesyltransferase and geranylgeranyltransferases I and II) catalyze the attachment of prenyl lipid anchors 15 or 20 carbons long to the carboxyl termini of a variety of eukaryotic proteins. Farnesyltransferase and geranylgeranyltransferase I both recognize a 'Ca₁a₂X' motif on their protein substrates; geranylgeranyltransferase II recognizes a different, non-CaaX motif. Each enzyme has two subunits. The genes encoding CaaX protein prenyltransferases are considerably longer than those encoding non-CaaX subunits, as a result of longer introns. Alternative splice forms are predicted to occur, but the extent to which each splice form is translated and the functions of the different resulting isoforms remain to be established. Farnesyltransferase-inhibitor drugs have been developed as anti-cancer agents and may also be able to treat several other diseases. The effects of these inhibitors are complicated, however, by the overlapping substrate specificities of geranylgeranyltransferase I and farnesyltransferase.     

Structures,mechanisms and inhibitors of undecaprenyl diphosphate synthase: A cis-prenyltransferase for bacterial peptidoglycan biosynthesis

Isoprenoids are an intensive group of compounds made from isopentenyl diphosphate (IPP), catalyzed by prenyltransferases such as farnesyl diphosphate (FPP) cyclases, squalene synthase, protein farnesyltransferases and geranylgeranyltransferases, aromatic prenyltransferases as well as a group of prenyltransferases (cis- and trans-types) catalyzing consecutive condensation reactions of FPP with specific numbers of IPP to generate linear products with designate chain lengths. These prenyltransferases play significant biological functions and some of them are drug targets. In this review, structures, mechanisms, and inhibitors of a cis-prenyltransferase, undecaprenyl diphosphate synthase (UPPS) that mediates bacterial peptidoglycan biosynthesis, are summarized for comparison with the most related trans-prenyltransferases and other prenyltransferases.