Regulation Of Sugar Transport And Metabolism In Oral Bacteria

口腔细菌中糖运输和代谢的调节

• 批准号: 7593354
• 负责人: John M Thompson
• 金额: $ 45.29万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 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/7593354
• 关键词: 4-nitrophenol; 6-Phospho-beta-glucosidase; 6-phospho-alpha-glucosidase; Acids; Active Sites; Amino Acids; Bacillus subtilis; Bacteria; Benign; Biochemistry; Catalysis; Class; Cleaved cell; Condition; Data; Dental caries; Deuterium; Energy-Generating Resources; Enzymes; Etiology; Family; Fermentation; Fusobacteria; Fusobacterium; Genetic; Gingivitis; Glucose; Glucosides; Glycoside Hydrolases; Glycosides; Growth; Hydrolase; Hydrolysis; Individual; Isotopes; Journals; Kinetics; Lactic acid; Left; Measurement; Measures; Metabolic; Metabolism; Mouth Diseases; NADH; Niacinamide; Object Attachment; Oral; Organism; Oxidation-Reduction; Pathogenicity; Pathway interactions; Pattern; Peer Review; Periodontitis; Property; Protons; Rate; Reaction; Regulation; Reporting; Series; Streptococcus; Sulfhydryl Compounds; Sulfides; Thermotoga maritima; chemical kinetics; cofactor; cytotoxic; deprotonation; inorganic phosphate; maltose 6-phosphate; microbial; oral bacteria; oral biofilm; oxidation; pathogen; sugar

GlvA, a 6-phospho-alpha-glucosidase from Bacillus subtilis, is assigned to glycoside hydrolase family 4. We have previously hypothesized that this unusual enzyme catalyzes the hydrolysis of maltose 6-phosphate via a redox-elimination-addition mechanism requiring NAD+ as cofactor. In the past year our studies have provided physico-chemical, and kinetic evidence to support the postulated hypothesis. In contrast to previous reports and consistent with the proposed mechanism, GlvA is only activated in the presence of the nicotinamide cofactor in its oxidized, and not the reduced NADH, form. Significantly, GlvA catalyzes the hydrolysis of both 6-phospho-alpha- and 6-phospho-beta-glucosides containing activated leaving groups such as p-nitrophenol and does so with retention and inversion, respectively, of anomeric configuration. Mechanistic details of the individual bond cleaving and forming steps were probed using a series of 6-phospho-alpha- and 6-phospho-beta-glucosides. Primary deuterium kinetic isotope effects (KIEs) were measured for both classes of substrates in which either the C2 or the C3 protons have been substituted with a deuterium atom, consistent with C-H bond cleavage at each center being partially rate -limiting. Kinetic parameters were also determined for 1-2H-substituted substrates, and depending on the substrates and the reaction conditions, the measurements of kcat and kcat/KM produced either no KIEs or inverse KIEs. In conjunction with results of Bronsted analyses with both aryl 6-phospho-alpha- and beta-glucosides, the kinetic data suggest that GlvA utilizes an E1cb mechanism analogous to that proposed for the Thermotoga maritima BglT, a 6-phospho-beta-glucosidase that is also included in family 4 of the glycosyl hydrolase superfamily (Yip, V.L.Y et al. (2006) Biochemistry 45, 571-580). The pattern of isotope effects measured, and the observation of very similar kcat values for all substrates including unactivated and natural substrates, indicate that the oxidation and deprotonation steps are rate-limiting steps in essentially all cases. This catalytic mechanism permits the cleavage of both alpha- and beta-glycosides within the same active site motif and, for activated substrates that do not require acid catalysis for cleavage, within the same active site. Remarkably, the sugar-6-phosphate product (glucose-6P), has the same anomeric (alpha) form in the two cases. A summary of our findings has recently appeared in the peer-reviewed journal, Biochemistry.

GlvA 是一种来自枯草芽孢杆菌的 6-磷酸-α-葡萄糖苷酶，属于糖苷水解酶家族 4。我们之前假设这种不寻常的酶通过需要 NAD+ 作为辅因子的氧化还原-消除-添加机制催化麦芽糖 6-磷酸的水解。在过去的一年中，我们的研究提供了物理化学和动力学证据来支持假设的假设。与之前的报道相反，并且与所提出的机制一致，GlvA 仅在烟酰胺辅因子以其氧化形式而不是还原的 NADH 形式存在时才被激活。值得注意的是，GlvA 催化含有活化离去基团（如对硝基苯酚）的 6-磷酸-α-和 6-磷酸-β-葡萄糖苷的水解，并分别保留和反转异头构型。使用一系列 6-磷酸-α-和 6-磷酸-β-葡萄糖苷探讨了各个键断裂和形成步骤的机制细节。测量了两类底物的初级氘动力学同位素效应（KIE），其中C2或C3质子已被氘原子取代，这与每个中心处的C-H键裂解部分限速一致。还确定了 1-2H 取代底物的动力学参数，并且根据底物和反应条件，kcat 和 kcat/KM 的测量结果要么不产生 KIE，要么产生反 KIE。结合芳基 6-磷酸-α-和 β-葡萄糖苷的布朗斯台德分析结果，动力学数据表明 GlvA 利用 E1cb 机制，类似于为海栖热袍菌 BglT（一种 6-磷酸-β-葡萄糖苷酶）提出的机制。也包括在糖基水解酶超家族的家族 4 中（Yip, V.L.Y 等人 (2006) Biochemistry 45， 571-580）。测量的同位素效应模式以及对所有底物（包括未活化和天然底物）非常相似的 kcat 值的观察表明，氧化和去质子化步骤基本上在所有情况下都是限速步骤。这种催化机制允许在同一活性位点基序内裂解α-和β-糖苷，并且对于不需要酸催化裂解的活化底物，在同一活性位点内裂解。值得注意的是，6-磷酸糖产物（葡萄糖-6P）在两种情况下具有相同的异头（α）形式。我们的研究结果摘要最近发表在同行评审期刊《生物化学》上。