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。与枯草芽孢杆菌类似，粪肠球菌通过磷酸烯醇丙酮酸（PEP）运输和磷酸化麦芽糖：麦芽糖磷酸转移酶系统（PTS）。麦芽糖 - 特异性PTS渗透酶由基因麦芽编码。然而，粪肠球菌缺乏编码6磷-Alpha-葡萄糖苷酶的MALA基因，该基因在枯草芽孢杆菌水解中麦芽糖-6-P中的葡萄糖和葡萄糖-6-P中的麦芽糖水解中。取而代之的是，编码麦芽糖磷酸化酶（MALP），磷酸葡萄糖酶和mutarotase的操纵子从麦芽启动。建议将MALP分解为麦芽糖-6-P中的葡萄糖-1-P和葡萄糖-6-P。但是，纯化的MALP磷脂麦芽糖，而不是麦芽糖-6-P。我们发现，麦芽的下游基因编码一种新型酶（MAPP），该酶脱磷酸化麦芽糖-6-P由PTS形成。通过MALP水解产生的细胞内麦芽糖将其水解为葡萄糖和葡萄糖1-P。麦芽糖通过麦芽糊精ABC转运蛋白慢慢摄取仅使MAPP的生长差，而不是MALP突变体。枯草芽孢杆菌突变体中MAPP的合成，积累了麦芽糖-6-P恢复麦芽糖的生长。 MAPP催化细胞内麦芽糖-6-P的去磷酸化，并通过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。多种磷酸化底物的去磷酸化测定表明，MAPP最好是脱磷酸化，含有O-Alpha-糖基链接的二糖。这些发现已在分子微生物学上发表。肺炎链球菌Tigr4的磷酸β-糖苷酶（BGLA-2）的结构和功能。肺炎链球菌是急性肺炎，中耳炎，脑膜炎和败血病的主要病因，每年导致全球数百万人死亡。在人类宿主中，肺炎链球菌遇到了各种糖缀合物，包括粘蛋白，宿主防御分子和上皮细胞上表面暴露的聚糖。与其他致病性微生物共同，肺炎链球菌会产生多种分泌或表面相关的糖苷酶，其功能包括宿主糖偶联物的修饰和水解。基因组测序与新毒力因子的探索结合，表明大量糖苷酶对于最大程度的肺炎链球菌是必需的。 BGLA-2由肺炎链球菌Tigr4的染色体DNA中的基因SP_0578编码。基因在高表达载体中克隆后，将BGLA-2（471个残基，MW 54,361）纯化为电泳同质性。磷酸β-葡萄糖苷酶（BGLA-2）的天然底物包括：6-磷酸纤维二糖，Gentiobiose-6p，Arbutin-6p，Arbutin-6p，盐酸盐-6p和相关的O-beta链接的二糖类磷酸盐。这些新型化合物的使用允许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-beta-葡萄糖苷酶BGLA-2（EC 3.2.1.86）催化β-1,4-链接纤维素-6-磷酸的水解以产生葡萄糖和葡萄糖-6-磷酸盐（G6P）。两种反应产物通过能量产生的糖酵解途径进一步代谢。在这项研究中，我们在2.0和2.4 Angstrom分辨率下介绍了BGLA-2的Apo和复合形式的第一个晶体结构（BGLA-2的非代谢类似物）。与其他GH-1酶类似，肺炎链球菌的BGLA-2的整体结构采用了典型的（β/alpha）8 tim-barrel，活性位点位于β桶的凸表面的中心。结构分析与从位置定位的突变蛋白获得的酶促数据结合使用，表明在BGLA-2的子站点+1处的三个芳族残基：Tyr126，Tyr303和Trp338，确定相对于（1,4）链接的6-磷酸苯甲酸-甲基葡聚糖底物的底物特异性。此外，发现其他三个残基：BGLA-2的Ser424，Lys430和Tyr432在水解选择性中对磷酸化而不是非磷酸化化合物起着重要作用。 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.我们的发现已在《生物化学杂志》中报道。枯草芽孢杆菌的LPLD是分配给糖苷水解酶家族4的α-半乳糖苷苷酶4。在对201 GH4酶的较早的系统发育分析中，我们注意到了一组具有基序CHEV的未知催化活性的蛋白质。 2008年报道了其中一种蛋白质的结构，枯草芽孢杆菌菌株168的LPLD，但尚未确定酶促活性。在过去的一年中，与美国和加拿大的研究人员合作，我们表明含有CHEV基序的蛋白质是α-半乳糖醛酸苷酶（S），其天然底物为alpha-1,4-di-galacturonate（galua2）。从本调查中获得的结果发表在FEBS Letters中。

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）

Metabolism of sugars by genetically diverse species of oral Leptotrichia.

DOI：
10.1111/j.2041-1014.2011.00627.x
发表时间：
2012-02
期刊：
Molecular oral microbiology
影响因子：
3.7
作者：
Thompson J;Pikis A
通讯作者：
Pikis A

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

DOI：
10.1111/mmi.12183
发表时间：
2013-04
期刊：
Molecular microbiology
影响因子：
3.6
作者：
Mokhtari A;Blancato VS;Repizo GD;Henry C;Pikis A;Bourand A;de Fátima Álvarez M;Immel S;Mechakra-Maza A;Hartke A;Thompson J;Magni C;Deutscher J
通讯作者：
Deutscher J

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

DOI：
10.1016/j.febslet.2013.02.004
发表时间：
2013-03-18
期刊：
FEBS letters
影响因子：
3.5
作者：
Thompson J;Pikis A;Rich J;Hall BG;Withers SG
通讯作者：
Withers SG

The gene CBO0515 from Clostridium botulinum strain Hall A encodes the rare enzyme N5-(carboxyethyl) ornithine synthase, EC 1.5.1.24.

来自肉毒杆菌 Hall A 菌株的基因 CBO0515 编码稀有酶 N5-（羧乙基）鸟氨酸合酶，EC 1.5.1.24。

DOI：
10.1128/jb.01044-09
发表时间：
2010
期刊：
Journal of bacteriology
影响因子：
3.2
作者：
Thompson,John;Hill,KarenK;Smith,TheresaJ;Pikis,Andreas
通讯作者：
Pikis,Andreas

Structural insights into the substrate specificity of a 6-phospho-β-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4.

肺炎链球菌 TIGR4 的 6-磷酸-β-葡萄糖苷酶 BglA-2 的底物特异性的结构见解。

DOI：
10.1074/jbc.m113.454751
发表时间：
2013
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Yu,Wei-Li;Jiang,Yong-Liang;Pikis,Andreas;Cheng,Wang;Bai,Xiao-Hui;Ren,Yan-Min;Thompson,John;Zhou,Cong-Zhao;Chen,Yuxing
通讯作者：
Chen,Yuxing