几丁质酶的三级结构和催化机制

几丁质酶广泛存在于各种生物中 ,包括含有其底物 (几丁质 )的真菌、昆虫等生物中 ,也存在于不含几丁质的植物中。因而它在各种生物中有不同的作用。细菌产生几丁质酶可以分解几丁质物质而获得营养 ;真菌中几丁质酶在其细胞分裂和形态建成等方面发挥着作用 ;昆虫等节肢动物中几丁质酶在它们的蜕皮等生长发育中起着重要作用 ;植物以及高等动物的几丁质酶可能主要在于防御病原物的侵染。近来 ,由于几丁质酶在抗真菌中的作用而备受关注。几丁质酶根据其氨基酸序列的同源性被分成 1 8和 1 9两个家族。1 8家族广泛存在于各种生物中 ,而 1 9家族大…     

氢酶结构及催化机理研究进展

氢酶是一类催化氢的氧化或质子还原的酶,它在微生物产氢过程中扮演着重要角色。根据氢酶所含的金属元素,可分为NiFe_氢酶、Fe-氢酶和不含金属元素的metal_free氢酶。大多数氢酶含有金属原子,它们参与氢酶活性中心和[Fe_S]簇的形成。氢酶的活性中心直接催化氢的氧化与质子的还原,[Fe_S]簇则参与氢酶催化过程中电子的传输。目前已有数种NiFe_氢酶和Fe_氢酶的X射线衍射晶体结构被阐明。根据metal_free氢酶的序列特征,推断其结构与NiFe_氢酶和Fe_氢酶之间存在较大差异。对氢酶活性中心和[Fe_S]簇的深入研究,揭示了氢酶催化反应的机理。     

植物几丁质酶的结构,基因及其表达

几丁质酶按其蛋白氨基酸序列结构的特征及同源性可分为六类,即：ＣｌａｓｓⅠ－Ⅵ。ＣｌａｓｓＩ在蛋白氨基酸结构上包括三个功能区域,Ｎ－端是富含半胱氨酸的几丁质结合工,约４０个氨基酸;Ｃ－端是酶的催化区,也是酶的主要功能区域,约３００个氨基酸;二者通过一个多变的交联区连接在一起。ＣｌａｓｓⅡ仅具有类似于ＣｌａｓｓⅠ的酶催化区域,而没有几丁质结构区和交联区。ＣｌａｓｓⅢ几丁质酶在氨基酸序列上与ＣｌａｓｓⅠ     

杆状病毒几丁质酶基因结构与功能的研究进展

杆状病毒几丁质酶基因是杆状病毒的非必需基因,是高度保守的基因。该基因在杆状病毒复制晚期表达产生几丁质酶,该酶N端具信号肽,中部是酶的活性区,C端是酶的内质网结合区。杆状病毒几丁质酶同时具有内切和外切几丁质酶活性,主要功能是水解昆虫体内的组成型几丁质。杆状病毒几丁质酶对于虫体液化是必需的,同时它还是原组织蛋白酶(pro-V-Cath)的分子伴侣,并与病毒侵染机制相关联。杆状病毒的几丁质酶基因与细菌的几丁质酶基因可能源于共同的祖先。     

植物几丁质酶的结构与功能、分类及进化

近年来对几丁质酶的研究越来越深入,资料也愈来愈多,有的植物几丁质酶除具有几丁质酶活性,还具有其它的活性,典型的几丁质酶由－N－端信号区,催化区和C－端延伸区组成,有的还有几丁质结合域,各项能域具有各自的功能,对植物几丁质酶的分类已经过多次改进,目前公认的分成4组9个亚组,有证据表明植物几丁质酶在进化过程中有遗传转座现象,但具有进化过程还有待进一步确证,对几丁质酶与其它一些蛋白的关系的了解有助于理解几丁质酶的起源和进化,由于几丁质酶具有独特的抗真菌特性,因而几丁质酶基因成为目前抗真菌基因工程研究的热之一。     

小型核酶的结构和催化机理

自然界存在的小型核酶主要有锤头型核酶、发夹型核酶、肝炎δ病毒（HDV）核酶和VS核酶.锤头型核酶由3个短螺旋和1个广义保守的连接序列组成;发夹型核酶的催化中心由两个肩并肩挨着的区域构成;HDV核酶折叠成包含五个螺旋臂（P1～P4）的双结结构;VS核酶由五个螺旋结构组成,这些螺旋结构通过两个连接域连接起来.小型核酶的催化机理与其分子结构密切相关.金属离子或特定碱基都可作为催化反应的关键成分.锤头型核酶的催化必须有金属离子（尤其是二价金属离子）参与,而发夹型核酶则完全不需要金属离子.基因组HDV核酶进行催化时要有金属离子和特定碱基互相配合.     

水稻几丁质酶基因克隆RCH8的DNA结构分析

用ＤＮＡ外切核酸酶Ⅲ和Ｓ１核酸酶生成连缺失突变体,用Ｓａｎｇｅｒ双脱氧链终止法对该克隆进行双向ＤＮＡ顺序测定,测序全长２０４９个碱基,初步确定了１０５７ｂｐ的５‘端上游顺序,９６６ｂｐ不含内含子的完整编码区和可能的ＴＡＴＡｂｏｘ等。所编码的３２２个氨基酸包括Ｎ－端２０个氨基酸的信号肽,其后４０个氨基酸长度的含８个半胱氨的ｈｅｖｅｉｎｘ结构域和一个催化结构域。     

植物几丁质酶的研究进展

几丁质酶是植物抗真菌基因工程的热点之一。本文叙述了植物几丁质酶的特性、结构和功能、基因结构;按最新资料对以前的植物几丁质酶的分类系统进行了完善;概述了几丁质酶的分子进化的各家观点及其模型,并归纳了植物几丁质酶的生物学作用。     

几丁质酶及其研究进展

本文从几丁质酶的分布、发育调节、可诱导性、分子生物学及抗病基因工程等方面近年来的进展进行了综合论述,并对其进一步的应用提出展望。     

ATP合酶的结构与催化机理

ATP合酶 (F1Fo 复合物) 是生物体内进行氧化磷酸化和光合磷酸化的关键酶.随着核磁共振、X射线晶体衍射、遗传学、化学交联等技术在ATP合酶研究中的广泛应用,ATP合酶的整体结构及其各组成亚基结构的研究都有很大的进展.其中细菌ATP合酶结构的研究更为深入.目前对质子通过Fo的转运方式提出两种模型:单通道和双半通道模型.对扭力矩的形成以及旋转催化也有了进一步的认识.Boyer提出的结合改变机理推动了ATP合酶催化机制的研究,现在主要有两点催化机制和三点催化机制.ATP合酶的催化反应受酶的构象变化和外在条件的调节.     

大分子核酶的结构和催化机制

自然界存在的大分子核酶主要有第一类内含子,第二类内含子,RNase P的RNA亚基。它们的结构非常复杂。且都需要金属离子参加催化反应。     

膜蛋白Presenilin 1的跨膜结构及催化机制

膜蛋白presenilin 1(PS1)是γ分泌酶的催化组分,是催化产生β淀粉样蛋白（β-amyloid,Aβ）的关键蛋白酶,因此也是治疗阿尔茨海默病（Alzheimer’s disease,AD）的主要靶点.PS1属于膜内裂解蛋白酶家族,这是一类在膜脂双层内部催化肽键水解断裂的蛋白酶.PS1其独特的跨膜结构和催化机制虽然还未完全揭示,但近期相关的研究取得了重要成果：PS1有10个疏水区,跨膜9次,其N端位于胞内,C端位于胞膜外或者内质网腔内,亦或不同程度地插入膜内,2个起催化作用的天冬氨酸残基都位于疏水性的膜内,膜蛋白底物被催化水解时必须先结合到酶的疏水表面上来,然后再进入位于活性部位.虽然PS1的晶体从未获得,但2006年首次解析的膜内裂解蛋白酶GlpG的晶体结构和所提出的催化机理为PS1催化机理的揭示奠定了基础,也为设计和筛选PS1/γ分泌酶的特异性抑制剂提供了理论依据.     

Crystal Structure and Catalytic Mechanism of 4-Methylmuconolactone Methylisomerase

When methyl-substituted aromatic compounds are degraded via ortho (intradiol)-cleavage of 4-methylcatechol, the dead-end metabolite 4-methylmuconolactone (4-ML) is formed. Degradation of 4-ML has only been described in few bacterial species, including Pseudomonas reinekei MT1. The isomerization of 4-ML to 3-methylmuconolactone (3-ML) is the first step required for the mineralization of 4-ML and is catalyzed by an enzyme termed 4-methylmuconolactone methylisomerase (MLMI). We identified the gene encoding MLMI in P. reinekei MT1 and solved the crystal structures of MLMI in complex with 3-ML at 1.4-Å resolution, with 4-ML at 1.9-Å resolution and with a MES buffer molecule at 1.45-Å resolution. MLMI exhibits a ferredoxin-like fold and assembles as a tight functional homodimeric complex. We were able to assign the active site clefts of MLMI from P. reinekei MT1 and of the homologous MLMI from Cupriavidus necator JMP134, which has previously been crystallized in a structural genomics project. Kinetic and structural analysis of wild-type MLMI and variants created by site-directed mutagenesis indicate Tyr-39 and His-26 to be the most probable catalytic residues. The previously proposed involvement of Cys-67 in covalent catalysis can now be excluded. Residue His-52 was found to be important for substrate affinity, with only marginal effect on catalytic activity. Based on these results, a novel catalytic mechanism for the isomerization of 4-ML to 3-ML by MLMI, involving a bislactonic intermediate, is proposed. This broadens the knowledge about the diverse group of proteins exhibiting a ferredoxin-like fold.     

Structure and Catalytic Mechanism of Yeast 4-Amino-4-deoxychorismate Lyase

Saccharomyces cerevisiae Abz2 is a pyridoxal 5′-phosphate (PLP)-dependent lyase that converts 4-amino-4-deoxychorismate (ADC) to para-aminobenzoate and pyruvate. To investigate the catalytic mechanism, we determined the 1.9 Å resolution crystal structure of Abz2 complexed with PLP, representing the first eukaryotic ADC lyase structure. Unlike Escherichia coli ADC lyase, whose dimerization is critical to the formation of the active site, the overall structure of Abz2 displays as a monomer of two domains. At the interdomain cleft, a molecule of cofactor PLP forms a Schiff base with residue Lys-251. Computational simulations defined a basic clamp to orientate the substrate ADC in a proper pose, which was validated by site-directed mutageneses combined with enzymatic activity assays. Altogether, we propose a putative catalytic mechanism of a unique class of monomeric ADC lyases led by yeast Abz2.     

Structure and Catalytic Mechanism of the Thioesterase CalE7 in Enediyne Biosynthesis

The biosynthesis of the enediyne moiety of the antitumor natural product calicheamicin involves an iterative polyketide synthase (CalE8) and other ancillary enzymes. In the proposed mechanism for the early stage of 10-membered enediyne biosynthesis, CalE8 produces a carbonyl-conjugated polyene with the assistance of a putative thioesterase (CalE7). We have determined the x-ray crystal structure of CalE7 and found that the subunit adopts a hotdog fold with an elongated and kinked substrate-binding channel embedded between two subunits. The 1.75-Å crystal structure revealed that CalE7 does not contain a critical catalytic residue (Glu or Asp) conserved in other hotdog fold thioesterases. Based on biochemical and site-directed mutagenesis studies, we proposed a catalytic mechanism in which the conserved Arg³⁷ plays a crucial role in the hydrolysis of the thioester bond, and that Tyr²⁹ and a hydrogen-bonded water network assist the decarboxylation of the β-ketocarboxylic acid intermediate. Moreover, computational docking suggested that the substrate-binding channel binds a polyene substrate that contains a single cis double bond at the C4/C5 position, raising the possibility that the C4=C5 double bond in the enediyne moiety could be generated by the iterative polyketide synthase. Together, the results revealed a hotdog fold thioesterase distinct from the common type I and type II thioesterases associated with polyketide biosynthesis and provided interesting insight into the enediyne biosynthetic mechanism.Enediyne natural products represent a family of structurally unique secondary metabolites with potent antitumor and antibiotic activities. Based on the structure of the bicyclic enediyne core, enediyne natural products are categorized into two groups with either a 9- or 10-membered enediyne moiety (1, 2). The antitumor activity of enediyne natural products derives from their capacity to induce chromosomal DNA cleavage through an oxidative radical mechanism (3). The biosynthetic mechanism for the enediyne moiety has been, however, elusive despite clues gleaned from early isotope-feeding experiments (4, 5). Pioneering genetic studies of the biosynthesis of calicheamicin and C-1027 from two research groups yielded major insights into the biosynthetic pathways, suggesting that an iterative polyketide synthase (PKS)5 plays a central role in the assembly of both the 9- and 10-membered enediyne moieties (6, 7). The gene clusters also contain open reading frames encoding hypothetical proteins for the downstream processing of the PKS product. The involvement of similar genes in enediyne biosynthesis was later confirmed for neocarzinostatin, maduropeptin, dynemicin, and several putative enediyne natural products in soil and marine microorganisms (8–11). Recently, based on the study on the 9-membered enediyne-containing C-1027, Shen and coworkers found that the iterative PKS (SgcE) and the putative thioesterase (SgcE10) generated a conjugated polyene (1,3,5,7,9,11,13-pentadecaheptaene) through an ACP-tethered 3-hydroxy-4,6,8,10,12,14-hexadecahexaene intermediate during co-expression in Escherichia coli (12). The release of the product catalyzed by the putative thioesterase SgcE10 presumably occurs through a combination of hydrolysis, decarboxylation, and dehydration steps. Recent biochemical studies of the iterative PKS (CalE8) from the biosynthetic pathway of calicheamicin also provided insight into the early steps of 10-membered enediyne biosynthesis (13, 14). It was observed that CalE8 produced a linear carbonyl-conjugated polyene (3,5,7,9,11,13-pentadecen-2-one (1)) with the assistance of the putative thioesterase CalE7 (Fig. 1). The putative biosynthetic intermediate 1 was proposed to derive from a 16-carbon-long β-ketocarboxylic intermediate tethered to CalE8 (13). Given the loss of one carbon unit during product release, a decarboxylation process was speculated to occur following the hydrolysis of the thioester bond.Open in a separate windowFIGURE 1.Calicheamicin and its biosynthesis. A, structure of calicheamicin γ′₁ with the incorporated acetate units in the 10-membered enediyne moiety highlighted in bold sticks. B, early steps of the biosynthetic pathway of the 10-membered enediyne as proposed by Kong et al. (13). The incorporated acetate units are highlighted in bold sticks with the configuration of the double bonds in the intermediates arbitrarily assigned. (AT, acyl transferase; KS, ketoacyl synthase; ACP, acyl carrier protein; KR, ketoreductase; DH, dehydratase; and PPTase, phosphopantetheinyl transferase.).Polyketide and non-ribosomal peptide synthesis generally involves the so-called type I and type II thioesterases for the release of final product or removal of aberrant products. Type I thioesterases (TE I) are cis-acting domains fused to the C terminus of the most downstream module of PKS or non-ribosomal peptide synthase for the release and cyclization of the final product (15, 16). By contrast, type II thioesterases (TE II) are discrete proteins responsible for the trans hydrolytic release of aberrant products (17–19). TE II proteins are structurally and evolutionarily related to a family of well known α/β hydrolase that contain 240–260 residues (20). A common serine esterase motif GXSXG and another downstream motif GXH are conserved in TE II proteins (21, 22). The stand-alone 146-amino acid-containing CalE7 does not belong to the TE II family, because it is neither an α/β fold hydrolase nor a protein containing the two conserved motifs for TE II. Instead, CalE7 shares moderate sequence homology with a family of hotdog fold proteins characterized by a long central α-helix packed against a five-stranded anti-parallel β-sheet. Such hotdog fold proteins include many characterized and hypothetical thioesterases that use acyl CoA as substrates (23). The three-dimensional structure and substrate specificity of several hotdog fold thioesterases have been determined, including YbgC from Helicobacter pylori (24), Paal from E. coli (25), HB8 from Thermos thermophilis (26), FcoT from Mycobacterium tuberculosis (27), YciA from Haemophilus influenzae (28), human THEM2 (25) and 4-hydroxylbenzoyl-CoA thioesterases (4-HBT) from Pseudomonas sp. Strain CBS and Arthrobacter sp. strain SU (29–31). Despite their diverse specificity toward acyl substrates (23, 25), all known hotdog fold thioesterases catalyze the hydrolysis of thioester bond using a Glu/Asp residue as nucleophile or general-base catalyst with the exception of FcoT (27). Here we present structural and biochemical data showing that CalE7 does not contain an acidic residue in its active site and is thus likely to utilize a different catalytic mechanism. The results also suggest that CalE7 facilitates a subsequent decarboxylation step to yield the carbonyl-conjugated polyene (1). Hence, the results introduce a hotdog fold thioesterase with a novel product-releasing mechanism in comparison with the traditional type I and II thioesterases associated with the biosynthesis of polyketide natural products. Furthermore, the crystal structure revealed a kinked substrate-binding channel that is predicted to bind a cis-double bond-containing polyene substrate, raising the possibility that CalE8 is able to generate a cis-double bond.     

Crystal Structure and Catalytic Mechanism of Leucoanthocyanidin Reductase from Vitis vinifera

Leucoanthocyanidin reductase (LAR) catalyzes the NADPH-dependent reduction of 2R,3S,4S-flavan-3,4-diols into 2R,3S-flavan-3-ols, a subfamily of flavonoids that is important for plant survival and for human nutrition. LAR1 from Vitis vinifera has been co-crystallized with or without NADPH and one of its natural products, (+)-catechin. Crystals diffract to a resolution between 1.75 and 2.72 Å. The coenzyme and substrate binding pocket is preformed in the apoprotein and not markedly altered upon NADPH binding. The structure of the abortive ternary complex, determined at a resolution of 2.28 Å, indicates the ordering of a short 3₁₀ helix associated with substrate binding and suggests that His122 and Lys140 act as acid-base catalysts. Based on our 3D structures, a two-step catalytic mechanism is proposed, in which a concerted dehydration precedes an NADPH-mediated hydride transfer at C4. The dehydration step involves a Lys-catalyzed deprotonation of the phenolic OH7 through a bridging water molecule and a His-catalyzed protonation of the benzylic hydroxyl at C4. The resulting quinone methide serves as an electrophilic target for hydride transfer at C4. LAR belongs to the short-chain dehydrogenase/reductase superfamily and to the PIP (pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase) family. Our data support the concept that all PIP enzymes reduce a quinone methide intermediate and that the major role of the only residue that has been conserved from the short-chain dehydrogenase/reductase catalytic triad (Ser…TyrXXXLys), that is, lysine, is to promote the formation of this intermediate by catalyzing the deprotonation of a phenolic hydroxyl. For some PIP enzymes, this lysine-catalyzed proton abstraction may be sufficient to trigger the extrusion of the leaving group, whereas in LAR, the extrusion of a hydroxide group requires a more sophisticated mechanism of concerted acid-base catalysis that involves histidine and takes advantage of the OH4, OH5, and OH7 substituents of leucoanthocyanidins.     

Three-dimensional Structure of Thermolysin

The electron density map of thermolysin indicates how the polypeptide chain is displaced throughout the overall shape of the molecule.