Regulation Of Sugar Transport And Metabolism In Lactic A

乳酸 A 中糖运输和代谢的调节

• 批准号: 6966394
• 负责人: john thompson
• 金额: --
• 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/6966394
• 关键词: Lactobacillaceae; Streptococcus mutans; alpha glucosidase; carbohydrate metabolism; carbohydrate transport; dental caries; isomer; oral bacteria; oxidation; sucrose

Previous studies in this laboratory pertaining to the mechanisms of transport and metabolism of sugars by microorganisms, led to the discovery of a large, but previously unrecognized family of glycosyl hydrolases (GH). These novel enzymes catalyze the cleavage of a wide variety of phosphorylated disaccharides including maltose-6?P, cellobiose-6?P and, most remarkably, the five phosphorylated isomers of sucrose. However, the characteristics that distinguish these hydrolases (designated Family GH4) from all others in the > 90 families comprising the Glycosyl Hydrolase superfamily, are their obligate requirements for NAD+, divalent metal ion and reducing conditions for activity. Whether these unique cofactors functioned in a catalytic or structural capacity was, until recently, unknown. However, our collaborations with international investigators in the past year, have provided the crystal structure of phospho - alpha - glucosidase (GlvA) from Bacillus subtilis in complex with its ligands to 2.05 Angstrom resolution. Analyses of the active site architecture, in conjunction with mechanistic studies and solvent isotope exchange, suggest a novel mechanism of glycoside hydrolysis requiring participation of both NAD(H) and Mn(2+) ion. The proposed four -step reaction involves hydride extraction at C3, and NAD+ mediated oxidation of the 3-OH group to a ketone. This oxidation step causes acidification of the C2 proton, and facilitates deprotonation by an enzymatic base. Thereafter, an acid -catalyzed reaction causes elimination of the glycosidic oxygen, and attendant formation of a 1,2 -unsaturated intermediate. This Michael-like acceptor undergoes base-catalyzed attack by water to generate the 3-keto form of glucose 6-phosphate (G6P). Finally, this keto - intermediate is reduced by the ?on-board? NADH to yield G6P, thereby completing the cycle, and returning the glycosyl hydrolase to its initial NAD/Mn(2+)-liganded active state. Sucrose is the precursor for glycan synthesis that facilitates attachment of oral pathogens eg., Streptococcus mutans to the tooth surface. Subsequent fermentation of this and other disaccharides (to lactic acid), initiates dental caries by promoting demineralization of tooth enamel. The belief that microorganisms are unable to metabolize the five isomers of sucrose, suggests the potential of these ?sweet? non-cariogenic compounds as substitutes for dietary sucrose in order to combat the etiology of dental caries. However, innovative studies conducted in the Microbial Biochemistry and Genetics Section have revealed rapid dissimilation of these isomers (trivially designated: trehalulose, turanose, maltulose, leucrose and palatinose) by several bacterial species including Fusobacteria, Klebsiella, Bacillus and Clostridia. Unique transport proteins and the NAD+/Mn(2+)-dependent phospho-alpha-glycosylhydrolases participate in the bacterial metabolism of sucrose isomers. The relevant genes have been cloned, sequenced, and proteins expressed for biochemical characterization. The absence of these genes in oral streptococci including S. mutans, explains the failure of these species to ferment the isomeric compounds. In view of the potential for inter-species transfer of genetic information (DNA), our studies suggest that caution be exercised in the widespread use of palatinose and leucrose as substitutes for dietary sucrose. Importantly, the determination of the solution-state conformations of the phosphorylated derivatives of sucrose and its isomers, together with our structural anlyses of Family 4 hydrolases, may permit the rational design of ?sucro-based? inhibitors for selective targeting of oral pathogens.

该实验室的先前研究与微生物的糖运输和代谢机制有关，导致发现了一个大型但未识别的糖基水解酶（GH）家族。这些新型酶催化了多种磷酸化的二糖的裂解，包括麦芽糖-6？p，cobobiose-6？p，最引人注目的是蔗糖的五个磷酸化异构体。但是，在包括糖基水解酶超家族的> 90个家庭中，将这些水解酶（指定的家族GH4）与其他所有其他家庭区分开的特征是他们对NAD+，二价金属离子和减少活性条件的义务要求。直到最近，这些独特的辅助因子是否以催化能力或结构能力发挥作用。但是，在过去的一年中，我们与国际研究人员的合作提供了磷-Alpha-葡萄糖苷酶（GLVA）的晶体结构，从枯草芽孢杆菌及其配体的配体及其配体分辨率为2.05 Angstrom分辨率。主动部位结构的分析以及机械研究和溶剂同位素交换，提出了一种新的糖苷水解机制，需要NAD（H）和MN（H）和MN（2+）离子的参与。提出的四个步骤反应涉及在C3处进行氢化物提取，而NAD+介导的3 -OH组氧化为酮。这种氧化步骤会导致C2质子的酸化，并通过酶碱促进去质子化。此后，酸催化的反应导致消除糖苷氧，并导致1,2不饱和中间体的随之而来的形成。这种类似迈克尔的受体经历了水的碱催化攻击，以产生3-酮形式的6-磷酸葡萄糖（G6P）。最后，这个酮 - 中级被板上减少了吗？ NADH产生G6P，从而完成周期，并将糖基水解酶返回其初始NAD/MN（2+） - 配体活性态。 蔗糖是聚糖合成的前体，可促进口服病原体的附着，例如链球菌突变体到牙齿表面。随后发酵这种和其他二糖（乳酸），通过促进牙釉质的脱矿化来引发龋齿。相信微生物无法代谢蔗糖的五个异构体，这表明这些潜力？甜？非钙化化合物作为饮食蔗糖的替代品，以对抗龋齿的病因。然而，在微生物生物化学和遗传学部分进行的创新研究表明，这些异构体（琐碎的指定：Trehalulose，turanose，maltulose，leucrose和palatinose）被包括梭状芽孢杆菌，Klebsiella，Klebsiella，Bacillbillus和Clostridia。独特的转运蛋白和NAD+/MN（2+） - 依赖性的磷酸化 - α-糖基水合酶参与蔗糖异构体的细菌代谢。相关基因已被克隆，测序和用于生化表征的蛋白质。包括链球菌的口服链球菌中的这些基因的不存在解释了这些物种发酵异构化合物的失败。鉴于遗传信息之间的种间转移（DNA）的潜力，我们的研究表明，在广泛使用palatinose和leucrose作为饮食中蔗糖的替代品时要谨慎行事。重要的是，确定了蔗糖及其异构体磷酸化衍生物的溶液状态构象，以及我们的族的结构厌食4个水解酶，是否可以允许基于Sucro的合理设计？选择性靶向口腔病原体的抑制剂。