Regulation Of Sugar Transport And Metabolism In Oral Bacteria

口腔细菌中糖运输和代谢的调节

基本信息

批准号：
8743727
负责人：
john thompson
金额：
$ 36.69万
依托单位：
NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8743727
关键词：
6-Phospho-beta-glucosidase 6-phospho-alpha-glucosidase ATP-Binding Cassette Transporters Accounting Active Sites Acute Pneumonia Adopted Alanine Algeria Alpha-galactosidase Alpha-glucosidase Amino Acid Motifs Arbutin Argentina Bacillus subtilis Biochemistry Biological Assay British Columbia Canada Catalysis Cellobiose Cessation of life Chemistry China Chinese People Collaborations Complex Cysteine DNA Data Databases Disaccharides Enterococcus faecalis Enzymes Epithelial Cells Exhibits Family France Genes Genetic Germany Glucose Glucose-6-Phosphate Glucosides Glycoside Hydrolases Goals Growth Host Defense Human Hydrolase Hydrolysis Investigation Ions Journals Kinetics Laboratories Letters Link Malt Grain Maltose Mediating Meningitis Metabolic Pathway Metabolism Methionine Microbe Microbiology Modification Molecular Mucins Muscle Form Glycogen Phosphorylase National Institute of Dental and Craniofacial Research Nucleotides Operon Otitis Media Pathway interactions Phosphoenolpyruvate Phosphoglucomutase Phosphorylase a Phosphorylases Phylogenetic Analysis Play Polysaccharides Property Protein Dephosphorylation Proteins Publishing Reaction Reducing Agents Regulation Reporting Research Research Personnel Resolution Role Science Septicemia Site Specificity Streptococcus pneumoniae Structural Biologist Structure Substrate Specificity Sulfur Surface System Technology Time Transferase Tryptophan Universities Virulence Virulence Factors alpha-glucuronidase analog beta Glucosidases beta barrel beta-Galactosidase beta-Glucosidase comparative divalent metal enzyme substrate expression vector gene cloning gene discovery genome sequencing gentiobiose inorganic phosphate interest maltodextrin member microbial microorganism mutant novel oral bacteria oxidation permease programs salicin sugar uptake

项目摘要

MALTOSE DISSIMILATION IN ENTEROCOCCUS FAECALIS. Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP): maltose phospho- transferase system (PTS). The maltose - specific PTS permease is encoded by the gene malT. However, E. faecalis lacks a malA gene encoding a 6-phospho-alpha-glucosidase which in B. subtilis hydrolyses maltose-6-P into glucose and glucose-6-P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose-6-P into glucose-1-P and glucose-6-P. However, purified MalP phosphorolyses maltose but not maltose-6-P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose-6-P formed by the PTS. The resulting intracellular maltose is hydrolyzed by MalP into glucose and glucose-1-P. Slow uptake of maltose via a maltodextrin ABC transporter allows poor growth only for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose-6-P restored growth on maltose. MapP catalyzes the dephosphorylation of intracellular maltose-6-P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose-1-P. MapP therefore connects PTS-mediated maltose uptake to maltose phosphorylase-catalyzed metabolism. Dephosphorylation assays with a wide variety of phosphorylated substrates revealed that MapP preferably dephosphorylates disaccharides containing an O-alpha-glycosyl linkage. These findings have been published in Molecular Microbiology. STRUCTURE AND FUNCTION OF PHOSPHO-BETA-GLUCOSIDASE (BGLA-2) FROM STREPTOCOCCUS PNEUMONIAE TIGR4. Streptococcus pneumonia is the major causative agent of acute pneumonia, otitis media, meningitis, and septicemia, which annually result in the deaths of millions worldwide. In the human host, S. pneumoniae encounters a variety of glyco-conjugates, including mucins, host defense molecules, and surface exposed glycans on epithelial cells. In common with other pathogenic microbes, S. pneumonia produces a variety of secreted or surface-associated glycosidases whose function(s) include the modification and hydrolysis of host glyco-conjugates. Genome sequencing, in combination with exploration of new virulence factors, suggests that a large number of glycosidases are necessary for maximum virulence of S. pneumoniae. BglA-2 is encoded by the gene Sp_0578 in the chromosomal DNA of S. pneumoniae TIGR4. After cloning of the gene in a high expression vector, BglA-2 (471 residues, MW 54,361) was purified to electrophoretic homogeneity. The natural substrates of phospho-beta-glucosidase (BglA-2) include: cellobiose-6-phosphate, gentiobiose-6P, arbutin-6P, salicin-6P and related O-beta-linked disaccharide phosphates. Use of these novel compounds permitted substrate specificity and kinetic analyses to be conducted. The 6-phospho-beta-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of beta-1,4-linked cellobiose -6-phosphate to yield glucose and glucose-6-phosphate (G6P). Both reaction products are further metabolized by the energy - generating glycolytic pathway. In this study, we present the first crystal structures of the apo- and complex-forms of BglA-2 with thiocellobiose-6P (a non-metabolizable analog of cellobiose-6P) at 2.0 and 2.4 Angstrom resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from S. pneumoniae adopts a typical (beta/alpha)8 TIM-barrel, with the active site located at the center of the convex surface of the beta-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues: Tyr126, Tyr303 and Trp338 at subsite +1 of BglA-2, determine substrate specificity with respect to (1,4)-linked 6-phospho-beta-glucoside substrates. Moreover, three additional residues: Ser424, Lys430 and Tyr432 of BglA-2, were found to play important roles in the hydrolytic selectivity towards phosphorylated, rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite -1 may contribute to the catalytic and substrate differences, between the structurally similar enzymes 6-phospho-beta-galactosidase and 6-phospho-beta-glucosidase, assigned to Family GH-1 of the Glycoside Hydrolase superfamily. Our findings have been reported in the Journal of Biological Chemistry. LPLD OF B. SUBTILIS IS AN ALPHA-GALACTURONIDASE ASSIGNED TO GLYCOSIDE HYDROLASE FAMILY 4. In an earlier phylogenetic analysis of 201 GH4 enzymes, we noted a group of proteins of unknown catalytic activity with the motif CHEV. The structure of one of those proteins, LplD from Bacillus subtilis strain 168, was reported in 2008 but the enzymatic activity was not determined. In the past year, in collaboration with investigators in the USA and Canada, we have shown that proteins containing the CHEV motif are alpha-galacturonidase(s) whose natural substrate is alpha-1,4-di-galacturonate (GalUA2). The results obtained from this investigation were published in FEBS Letters.

粪肠球菌中的麦芽糖异化。与枯草芽孢杆菌相似，粪肠球菌通过磷酸烯醇丙酮酸（PEP）：麦芽糖磷酸转移酶系统（PTS）运输和磷酸化麦芽糖。麦芽糖特异性 PTS 通透酶由 malT 基因编码。然而，粪肠球菌缺乏编码6-磷酸-α-葡萄糖苷酶的malA基因，该酶在枯草芽孢杆菌中将麦芽糖-6-P水解成葡萄糖和葡萄糖-6-P。相反，编码麦芽糖磷酸化酶 (MalP)、磷酸葡萄糖变位酶和变旋酶的操纵子从 malT 的上游开始。 MalP被建议将麦芽糖-6-P分解成葡萄糖-1-P和葡萄糖-6-P。然而，纯化的 MalP 会磷酸化麦芽糖，但不会磷酸化 maltose-6-P。我们发现 malT 下游的基因编码一种新型酶 (MapP)，可以使 PTS 形成的 maltose-6-P 去磷酸化。产生的细胞内麦芽糖被 MalP 水解成葡萄糖和葡萄糖-1-P。通过麦芽糖糊精 ABC 转运蛋白缓慢摄取麦芽糖仅导致 mapP 生长不良，而 malP 突变体则不然。积累 maltose-6-P 的枯草芽孢杆菌突变体中 MapP 的合成恢复了麦芽糖的生长。 MapP 催化细胞内麦芽糖-6-P 去磷酸化，所得麦芽糖被枯草芽孢杆菌麦芽糖磷酸化酶转化为葡萄糖和葡萄糖-1-P。因此，MapP 将 PTS 介导的麦芽糖摄取与麦芽糖磷酸化酶催化的代谢联系起来。使用多种磷酸化底物进行的去磷酸化测定表明，MapP 优先对含有 O-α-糖基键的二糖进行去磷酸化。这些发现已发表在《分子微生物学》上。肺炎链球菌 TiGR4 的磷酸-β-葡萄糖苷酶 (BGLA-2) 的结构和功能。肺炎链球菌是急性肺炎、中耳炎、脑膜炎和败血症的主要病原体，每年导致全世界数百万人死亡。在人类宿主中，肺炎链球菌遇到多种糖缀合物，包括粘蛋白、宿主防御分子和上皮细胞上表面暴露的聚糖。与其他病原微生物一样，肺炎链球菌产生多种分泌型或表面相关糖苷酶，其功能包括修饰和水解宿主糖缀合物。基因组测序与新毒力因子的探索相结合表明，肺炎链球菌的最大毒力需要大量糖苷酶。 BglA-2 由肺炎链球菌 TIGR4 染色体 DNA 中的基因 Sp_0578 编码。将基因克隆到高表达载体中后，将 BglA-2（471 个残基，MW 54,361）纯化至电泳均质。磷酸-β-葡萄糖苷酶 (BglA-2) 的天然底物包括：纤维二糖-6-磷酸、龙胆二糖-6P、熊果苷-6P、水杨苷-6P 和相关的 O-β 连接二糖磷酸酯。使用这些新型化合物可以进行底物特异性和动力学分析。来自糖苷水解酶家族 1 (GH-1) 的 6-磷酸-β-葡萄糖苷酶 BglA-2 (EC 3.2.1.86) 催化 β-1,4-连接纤维二糖 -6-磷酸水解，产生葡萄糖和葡萄糖-6 -磷酸盐（G6P）。两种反应产物均通过产生能量的糖酵解途径进一步代谢。在这项研究中，我们分别以 2.0 和 2.4 埃的分辨率首次展示了 BglA-2 与 thiocellodiose-6P（纤维二糖-6P 的不可代谢类似物）的脱辅基形式和复合形式的晶体结构。与其他GH-1酶类似，肺炎链球菌BglA-2的整体结构采用典型的(β/α)8 TIM-桶，活性位点位于β-桶凸面的中心。结构分析结合从定点突变蛋白获得的酶数据表明，BglA-2 亚位点 +1 处的三个芳香族残基：Tyr126、Tyr303 和 Trp338，决定了 (1,4) 连接 6 的底物特异性。 -磷酸-β-葡萄糖苷底物。此外，发现BglA-2的另外三个残基：Ser424、Lys430和Tyr432在磷酸化而非非磷酸化化合物的水解选择性中发挥重要作用。比较结构分析表明，在结构相似的酶 6-磷酸-β-半乳糖苷酶和 6-磷酸-β-葡萄糖苷酶（分配给家族）之间，亚位点 -1 处的色氨酸与蛋氨酸/丙氨酸残基可能导致催化和底物差异。糖苷水解酶超家族的 GH-1。我们的研究结果已发表在《生物化学杂志》上。枯草芽孢杆菌的 LPLD 是一种属于糖苷水解酶家族 4 的 α-半乳糖醛酸酶。在对 201 种 GH4 酶的早期系统发育分析中，我们注意到一组具有 CHEV 基序的催化活性未知的蛋白质。其中一种蛋白质（来自枯草芽孢杆菌菌株 168 的 LplD）的结构于 2008 年被报道，但酶活性尚未测定。去年，我们与美国和加拿大的研究人员合作，证明含有 CHEV 基序的蛋白质是 α-半乳糖醛酸酶，其天然底物是 α-1,4-二-半乳糖醛酸 (GalUA2)。这项调查的结果发表在 FEBS Letters 上。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Structural insight into the catalytic mechanism of gluconate 5-dehydrogenase from Streptococcus suis: Crystal structures of the substrate-free and quaternary complex enzymes.

猪链球菌葡萄糖酸 5-脱氢酶催化机制的结构洞察：无底物和四元复合酶的晶体结构。

DOI：
发表时间：
2009-02
期刊：
影响因子：
0
作者：
Zhang, Qiangmin;Peng, Hao;Gao, Feng;Liu, Yiwei;Cheng, Hao;Thompson, John;Gao, George F
通讯作者：
Gao, George F

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose 6'-phosphate phosphatase (MapP).

粪肠球菌通过新型麦芽糖 6-磷酸磷酸酶 (MapP) 连接两条不相容的代谢途径来利用麦芽糖。

DOI：
发表时间：
2013-04
期刊：
影响因子：
3.6
作者：
Mokhtari, Abdelhamid;Blancato, Víctor S;Repizo, Guillermo D;Henry, Céline;Pikis, Andreas;Bourand, Alexa;de Fátima Álvarez, María;Immel, Stefan;Mechakra;Hartke, Axel;Thompson, John;Magni, Christian;Deutscher, Josef
通讯作者：
Deutscher, Josef

Evolution and biochemistry of family 4 glycosidases: implications for assigning enzyme function in sequence annotations.

家族 4 糖苷酶的进化和生物化学：在序列注释中分配酶功能的影响。

DOI：
发表时间：
2009-11
期刊：
影响因子：
10.7
作者：
Hall, Barry G;Pikis, Andreas;Thompson, John
通讯作者：
Thompson, John

Crystal structures of Streptococcus suis mannonate dehydratase (ManD) and its complex with substrate: genetic and biochemical evidence for a catalytic mechanism.

猪链球菌甘露酸脱水酶 (ManD) 及其底物复合物的晶体结构：催化机制的遗传和生化证据。

DOI：
发表时间：
2009-09
期刊：
影响因子：
3.2
作者：
Zhang, Qiangmin;Gao, Feng;Peng, Hao;Cheng, Hao;Liu, Yiwei;Tang, Jiaqi;Thompson, John;Wei, Guohua;Zhang, Jingren;Du, Yuguo;Yan, Jinghua;Gao, George F
通讯作者：
Gao, George F

α-Galacturonidase(s): a new class of Family 4 glycoside hydrolases with strict specificity and a unique CHEV active site motif.

α-半乳糖醛酸酶：一类新的家族 4 糖苷水解酶，具有严格的特异性和独特的 CHEV 活性位点基序。