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是枯草芽孢杆菌的6磷 - α-葡萄糖苷酶，分配给糖苷水解酶家族4。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，以支持假设的假设。与以前的报告相反，与所提出的机制一致，GLVA仅在烟酰胺辅因子的存在中被激活，而不是氧化的nADH形式减少。值得注意的是，GLVA催化6-磷酸α-和6-磷酸β-葡萄糖苷的水解含有激活的离开基团，例如p-硝基苯酚，并分别在保留和倒置的异常构型中进行。使用一系列6磷-Alpha-和6磷酸β-葡萄糖剂探测了单个键切割和形成步骤的机械细节。对于两类的底物测量了原发性氘动物同位素效应（KIE），其中C2或C3质子已用氘原子取代，与每个中心的C -H键裂解一致，是部分限制限制的。还确定了1-2H取代的底物的动力学参数，取决于底物和反应条件，KCAT和KCAT/KM的测量结果无需KIES或KIES。结合与芳基的6-磷酸α-和β-葡萄糖苷的布朗斯特德分析结果，动力学数据表明，GLVA利用了类似于Thermotoga Maritima bglt（6磷酸甘露酚）的E1CB机制，该机制也包括在6-磷酸中的水合物。 （Yip，V.L.Y等人（2006）生物化学45，571-580）。测量的同位素效应的模式以及所有底物（包括未激活和自然底物）非常相似的KCAT值的观察表明，在所有情况下，氧化和去质子化步骤是限制速率的步骤。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进行裂解的激活底物，在同一活性位点内。值得注意的是，在两种情况下，糖6-磷酸产物（葡萄糖-6p）具有相同的异常（alpha）形式。我们的发现的摘要最近出现在同行评审的日记生物化学中。