植物β-葡萄糖苷酶的研究进展

β-葡萄糖苷酶是一种糖苷水解酶,广泛存在于动物、植物和微生物中。β-葡萄糖苷酶能够水解非还原性末端糖基,在植物细胞壁代谢、植物激素激活以及逆境防御等方面发挥着重要作用。β-葡萄糖苷酶依据其氨基酸序列可以分为GH1、GH3、GH5、GH7、GH9、GH12、GH35、GH116等8个家族;但是,目前仅对GH1和GH3有较深入的研究,其他家族的功能依旧不清楚。综述了近年来植物中β-葡萄糖苷酶的结构、理化性质、底物特异性、催化机制以及糖苷水解酶家族在植物中的功能等方面的研究进展,总结了植物中β-葡萄糖苷酶研究中存在的问题,并指出今后的研究方向。     

酰胺酶的挖掘及在有机合成中的应用进展

酰胺酶是一类作用于分子内酰胺键,催化酰胺水解生成相应的羧酸和氨的重要生物催化剂。酰胺酶的广底物谱,高度化学、区域和立体选择性等特性,使其在制备医药及农用化学品等方面占有重要地位。根据氨基酸序列的差异,酰胺酶可分为酰胺酶标签家族和腈水解酶家族两大类。本文结合笔者研究团队在酰胺酶生物催化领域的研究工作,综述了近年来酰胺酶结构解析和催化机制等方面的研究进展,并介绍了酰胺酶在医药、农用化学品合成中的应用。     

微生物GH13家族淀粉脱支酶研究进展

普鲁兰酶和异淀粉酶都具有典型的(β/α)8桶状结构,属于GH13家族淀粉脱支酶.GH13家族的淀粉脱支酶能够专一、高效地水解淀粉分支部位的α-1,6-糖苷键,可以有效提高淀粉原料利用率和生产效率,在淀粉加工工业中具有重要的应用价值,因此近年来对GH13家族淀粉脱支酶的研究逐渐增多.本文系统地综述了微生物来源的GH13家族淀粉脱支酶的国内外研究进展,分别对普鲁兰酶和异淀粉酶的底物特异性及结构基础、研究现状以及应用和研究新趋势进行了阐述.并对GH13家族淀粉脱支酶研究中存在的问题和下一步开发方向提出了见解.     

植物类型III聚酮合酶超家族晶体结构与功能

植物类型Ⅲ聚酮化合物合酶(PKS)催化合成多种植物次生代谢产物的基本分子骨架,参与植物体许多重要生物学功能的行使,一直是研究蛋白结构与功能关系、基于结构进行分子改造的重要模式分子家族。目前在蛋白质数据库(PDB)中有超过80个不同种属来源的类型Ⅲ PKS的三维结构被报道,其中包括了研究最为透彻的查尔酮合酶在内的7种酶的晶体结构,这些结构的发表对于阐明该类酶复杂多变的底物专一性、链延伸和不同的环化反应机制奠定了结构基础。三维空间结构解析以及基于定点突变的结构功能分析是进行酶工程、基因工程的基础。以下系统综述了植物类型Ⅲ PKS超家族晶体结构和功能的研究进展。     

细菌几丁质酶结构、功能及分子设计的研究进展

几丁质是仅次于纤维素的第二大天然多糖，由N-乙酰-D-氨基葡萄糖聚合而成，具有重要的应用价值。自然界中几丁质可被细菌高效降解。细菌可分泌多种几丁质降解酶类，主要分布在GH18家族和GH19家族中。细菌中几丁质降解酶基因存在明显的基因扩增及多结构域组合现象，不同家族、不同作用模式的几丁质酶系协同作用打破复杂的抗降解屏障，完成结晶几丁质的高效降解。因此，深入分析细菌几丁质酶结构与功能，对几丁质高效降解与高值转化应用具有重要意义。本文介绍了细菌几丁质酶的分类、结构特点与催化作用机制；总结了不同细菌胞外几丁质降解酶系的协同降解模式；针对几丁质酶家族分子改造的研究进展，展望了以结构生物信息学及大数据深度学习为基础的蛋白质工程设计策略在今后改造中的作用，为几丁质酶的设计与理性改造提供新的视角与思路。     

降解木质纤维素放线菌的功能组学分析及工业应用前景

放线菌是一种高GC含量的革兰氏阳性细菌,在陆生、高温的木质纤维素降解生境中占据十分重要的地位.降解木质纤维素菌株的功能基因组分析发现降解纤维素的酶种类和数目相对较多,而降解半纤维素以及果胶成分的酶相对真菌较少.其中,降解纤维素的酶类主要以GH6家族外切酶为主,部分含有GH9和GH48家族的纤维素酶,基因组中还含有AA10家族的多糖裂解氧化酶,因此放线菌可通过持续性水解与氧化双重机制高效降解结晶纤维素.放线菌可通过双精氨酸转运系统快速将已正确折叠的降解酶类分泌至胞外,这些酶分子常具有多个功能结构域,具有耐高温、耐碱性以及高活力等特征.放线菌在木质纤维素降解及次级代谢产物等方面的特点与优势使得其具有巨大的工业应用前景.     

纤维素酶家族及其催化结构域分子改造的新进展

纤维素酶的分子改造是其催化性能改进及催化效率提升的重要手段。近年来,组学技术与结构测定技术的迅速发展,人们已建立了包括糖苷水解酶(Glycoside hydrolase,GH)在内的碳水化合物活性酶组分数据库。通过对同一蛋白家族进行序列比对、分子进化分析与祖先基因重构,以结构模建分析为指导的纤维素酶分子改造,可以明显缩小序列或结构的搜索空间,加快酶分子改造的速度,增大理性设计成功的概率;同时针对催化中心活性架构的分析可以进一步阐明纤维素酶的催化机理与酶分子持续性降解机制。文中主要对纤维素酶家族及其催化结构域的分子改造取得的最新进展作了综述。在后基因组时代基于蛋白质家族中的海量数据分析,以其保守结构信息为指导的理性设计,将会成为纤维素酶分子改造的重要方向,从而推动生物质转化工艺的快速发展。     

拮抗菌与病原菌碳水化合物酶类比较分析

拮抗菌通过分泌细胞壁降解酶降解病原菌细胞壁组分是其重要的拮抗方式。本研究以2个拮抗菌(绿色木霉和枯草芽孢杆菌)和7个植物病原菌为研究对象,对碳水化合物酶类CAZymes(Carbohydrate-active enzymes)进行了注释和比较分析,明确了拮抗菌相对于病原菌发生扩增的细胞壁降解酶亚家族;利用ClustalX2.0多序列比对、Mega4.0构建系统发育树和MEME软件预测保守基序的方法,分析了扩增亚家族的序列结构和进化特征。结果表明,拮抗菌中可能参与病原菌细胞壁降解酶类的亚家族CBM50、GH25和GH73发生了显著扩增,并通过序列比较和进化分析初步明确了扩增的亚家族与拮抗菌特异降解病原菌细胞壁组分之间存在关联,为拮抗菌细胞壁降解酶类亚家族CBM50、GH25和GH73参与拮抗的分子机理提供理论依据。     

罗汉果几丁质酶基因家族的生物信息学分析

几丁质酶是一类在植物抵抗病原真菌等过程中具有重要作用的蛋白质,为探讨几丁质酶在罗汉果抗根结线虫病中的调控作用,本研究基于南方根结线虫侵染下的罗汉果幼苗根系的转录组测序结果,采用生物信息学技术对筛选到的15个罗汉果几丁质酶基因进行分析。结果表明,15个罗汉果几丁质相关蛋白基因编码的氨基酸序列其N段均有一段信号肽,亚细胞定位在胞外;分子量从27 kDa到37 KDa不等;多数为酸性蛋白。基于氨基酸保守结构域和系统发育关系分析,15个罗汉果几丁质酶分属于GH18和GH19两大家族中的3个组别(Ⅰ、Ⅲ和Ⅳ)的成员,GH18家族成员三级结构预测具有典型的(α/β)_8桶状结构,而GH19家族成员三级结构预测只有α螺旋结构域。这些分析结果可为今后深入研究罗汉果几丁质酶的生物学功能和调控机制提供一定的理论依据,为罗汉果抗根结线虫病育种提供参考。     

CYP116B家族单加氧酶的发现、表征及分子改造研究进展

CYP116B家族单加氧酶属于细胞色素P450单加氧酶的第IV家族,能够催化包括羟化、硫醚氧化、O-脱烷基、N-脱烷基和环氧化等在内的多种类型反应,具有广阔的应用前景。近年来,多个CYP116B家族成员酶的发现、分子改造及底物谱拓展使人们对它的酶学性质有了更为深入的理解,为开发新型具有工业应用潜力的CYP116B家族成员酶提供了研究基础。本文中,笔者主要从CYP116B家族单加氧酶的发现、表征、分子改造及结构功能关系等方面综述CYP116B在生物催化领域的研究进展。     

Structural and functional basis for substrate specificity and catalysis of levan fructotransferase

Levan is β-2,6-linked polymeric fructose and serves as reserve carbohydrate in some plants and microorganisms. Mobilization of fructose is usually mediated by enzymes such as glycoside hydrolase (GH), typically releasing a monosaccharide as a product. The enzyme levan fructotransferase (LFTase) of the GH32 family catalyzes an intramolecular fructosyl transfer reaction and results in production of cyclic difructose dianhydride, thus exhibiting a novel substrate specificity. The mechanism by which LFTase carries out these functions via the structural fold conserved in the GH32 family is unknown. Here, we report the crystal structure of LFTase from Arthrobacter ureafaciens in apo form, as well as in complexes with sucrose and levanbiose, a difructosacchride with a β-2,6-glycosidic linkage. Despite the similarity of its two-domain structure to members of the GH32 family, LFTase contains an active site that accommodates a difructosaccharide using the -1 and -2 subsites. This feature is unique among GH32 proteins and is facilitated by small side chain residues in the loop region of a catalytic β-propeller N-domain, which is conserved in the LFTase family. An additional oligosaccharide-binding site was also characterized in the β-sandwich C-domain, supporting its role in carbohydrate recognition. Together with functional analysis, our data provide a molecular basis for the catalytic mechanism of LFTase and suggest functional variations from other GH32 family proteins, notwithstanding the conserved structural elements.     

Function and structure studies of GH family 31 and 97 α-glycosidases

A huge number of glycoside hydrolases are classified into the glycoside hydrolase family (GH family) based on their amino-acid sequence similarity. The glycoside hydrolases acting on α-glucosidic linkage are in GH family 4, 13, 15, 31, 63, 97, and 122. This review deals mainly with findings on GH family 31 and 97 enzymes. Research on two GH family 31 enzymes is described: clarification of the substrate recognition of Escherichia coli α-xylosidase, and glycosynthase derived from Schizosaccharomyces pombe α-glucosidase. GH family 97 is an aberrant GH family, containing inverting and retaining glycoside hydrolases. The inverting enzyme in GH family 97 displays significant similarity to retaining α-glycosidases, including GH family 97 retaining α-glycosidase, but the inverting enzyme has no catalytic nucleophile residue. It appears that a catalytic nucleophile has been eliminated during the molecular evolution in the same way as a man-made nucleophile mutant enzyme, which catalyzes the inverting reaction, as in glycosynthase and chemical rescue.     

Function and Structure Studies of GH Family 31 and 97 α-Glycosidases

A huge number of glycoside hydrolases are classified into the glycoside hydrolase family (GH family) based on their amino-acid sequence similarity. The glycoside hydrolases acting on α-glucosidic linkage are in GH family 4, 13, 15, 31, 63, 97, and 122. This review deals mainly with findings on GH family 31 and 97 enzymes. Research on two GH family 31 enzymes is described: clarification of the substrate recognition of Escherichia coli α-xylosidase, and glycosynthase derived from Schizosaccharomyces pombe α-glucosidase. GH family 97 is an aberrant GH family, containing inverting and retaining glycoside hydrolases. The inverting enzyme in GH family 97 displays significant similarity to retaining α-glycosidases, including GH family 97 retaining α-glycosidase, but the inverting enzyme has no catalytic nucleophile residue. It appears that a catalytic nucleophile has been eliminated during the molecular evolution in the same way as a man-made nucleophile mutant enzyme, which catalyzes the inverting reaction, as in glycosynthase and chemical rescue.     

Revisiting glycoside hydrolase family 20 β-N-acetyl-d-hexosaminidases: Crystal structures,physiological substrates and specific inhibitors

Glycoside hydrolase family 20 β-N-acetyl-d-hexosaminidases (GH20s) catalyze the hydrolysis of glycosidic linkages in glycans, glycoproteins and glycolipids. The diverse substrates of GH20s account for their various roles in many important bioprocesses, such as glycoprotein modification, glycoconjugate metabolism, gamete recognition and chitin degradation in fungal cell walls and arthropod exoskeletons. Defects in human GH20s cause lysosomal storage diseases, Alzheimer's disease and osteoarthritis. Similarly, lower levels of GH20s arrest arthropod molting. Although GH20s are promising targets for drug and agrochemical development, designing bioactive molecules to target one specific enzyme is challenging because GH20s share a conserved catalytic mechanism. With the development of structural biology, the last two decades have witnessed a dramatic increase in crystallographic investigations of liganded and unliganded GH20s, providing core information for rational molecular designs. This critical review summarizes recent research advances in GH20s, with a focus on their structural basis of substrate specificity as well as on inhibitor design. As more crystal structures of targeted GH20s are determined and analyzed, dynamics of their catalysis and inhibition will also be elucidated, which will facilitate the development of new drugs, pesticides and agrochemicals.     

Structural basis for inhibition of xyloglucan-specific endo-β-1,4-glucanase (XEG) by XEG-protein inhibitor

Microorganisms such as plant pathogens secrete glycoside hydrolases (GHs) to digest the polysaccharide chains of plant cell walls. The degradation of cell walls by these enzymes is a crucial step for nutrition and invasion. To protect the cell wall from these enzymes, plants secrete glycoside hydrolase inhibitor proteins (GHIPs). Xyloglucan-specific endo-β-1,4-glucanase (XEG), a member of GH family 12 (GH12), could be a great threat to many plants because xyloglucan is a major component of the cell wall in most plants. Understanding the inhibition mechanism of XEG by GHIP is therefore of great importance in the field of plant defense, but to date the mechanism and specificity of GHIPs remain unclear. We have determined the crystal structure of XEG in complex with extracellular dermal glycoprotein (EDGP), a carrot GHIP that inhibits XEG. The structure reveals that the conserved arginines of EDGP intrude into the active site of XEG and interact with the catalytic glutamates of the enzyme. We have also determined the crystal structure of the XEG-xyloglucan complex. These structures show that EDGP closely mimics the XEG-xyloglucan interaction. Although EDGP shares structural similarity to a wheat GHIP (Triticum aestivum xylanase inhibitor-IA (TAXI-IA)) that inhibits GH11 family xylanases, the arrangement of GH and GHIP in the XEG-EDGP complex is distinct from that in the xylanase-TAXI-IA complex. Our findings imply that plants have evolved structures of GHIPs to inhibit different GH family members that attack their cell walls.     

Microbe-derived non-necrotic glycoside hydrolase family 12 proteins act as immunogenic signatures triggering plant defenses

Plant pattern recognition receptors (PRRs) are sentinels at the cell surface sensing microbial invasion and activating innate immune responses. During infection, certain microbial apoplastic effectors can be recognized by plant PRRs, culminating in immune responses accompanied by cell death. However, the intricated relationships between the activation of immune responses and cell death are unclear. Here, we studied the glycoside hydrolase family 12 (GH12) protein, Ps109281, secreted by Phytophthora sojae into the plant apoplast during infection. Ps109281 exhibits xyloglucanase activity, and promotes P. sojae infection in a manner dependent on the enzyme activity. Ps109281 is recognized by the membrane-localized receptor-like protein RXEG1 and triggers immune responses in various plant species. Unlike other characterized GH12 members, Ps109281 fails to trigger cell death in plants. The loss of cell death induction activity is closely linked to a sequence polymorphism at the N-terminus. This sequence polymorphism does not affect the in planta interaction of Ps109281 with the recognition receptor RXEG1, indicating that cell death and immune response activation are determined using different regions of the GH12 proteins.Such GH12 protein also exists in other Phytophthora and fungal pathogens. Taken together, these results unravel the evolution of effector sequences underpinning different immune outputs.     

Characterization of a glycoside hydrolase family 42 ��-galactosidase from Deinococcus geothermalis

A putative recombinant β-galactosidase from Deinococcus geothermalis was purified as a single 79 kDa band of 42 U activity/mg using His-Trap affinity chromatography. The molecular mass of the native enzyme was a 158 kDa dimer. The catalytic residues E151 and E325 of β-galactosidase from D. geothermalis were conserved in all aligned GH family 42 β-galactosidases, indicating that this enzyme is also a GH family 42 β-galactosidase. Maximal activity of the enzyme was at pH 6.5 and 60°C. It has a unique hydrolytic activity for p-nitrophenyl(pNP)-β-D-galactopyranoside (k (cat)/K (m) = 69 s(-1) mM(-1)), pNP-β-D-fucopyranoside (13), oNP-β-D-galactopyranoside (9.5), oNP-β-D-fucopyranoside (2.6), lactose (0.97), and pNP-α-L-arabinopyranoside (0.78), whereas no activity, or less than 2% of the pNP-β-D-galactopyranoside activity, for other pNP- and oNP-glycosides.