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.

该实验室先前有关微生物糖转运和代谢机制的研究发现了一个大型但以前未被识别的糖基水解酶 (GH) 家族。这些新型酶催化多种磷酸化二糖的裂解，包括麦芽糖-6?P、纤维二糖-6?P，以及最引人注目的蔗糖的五种磷酸化异构体。然而，这些水解酶（命名为 GH4 家族）与糖基水解酶超家族超过 90 个家族中的所有其他酶的区别特征是它们对 NAD+、二价金属离子和活性还原条件的必然要求。直到最近，这些独特的辅助因子是否具有催化或结构功能尚不清楚。然而，我们去年与国际研究人员合作，提供了来自枯草芽孢杆菌的磷酸-α-葡萄糖苷酶 (GlvA) 及其配体复合物的晶体结构，分辨率为 2.05 埃。对活性位点结构的分析，结合机理研究和溶剂同位素交换，提出了一种需要 NAD(H) 和 Mn(2+) 离子参与的糖苷水解的新机制。所提出的四步反应涉及 C3 处的氢化物萃取，以及 NAD+ 介导的 3-OH 基团氧化为酮。该氧化步骤导致 C2 质子酸化，并促进酶碱的去质子化。此后，酸催化反应引起糖苷氧的消除，并伴随形成1,2-不饱和中间体。这种类似迈克尔的受体经历水的碱催化攻击，生成 3-酮形式的葡萄糖 6-磷酸 (G6P)。最后，该酮-中间体被“机载”还原。 NADH 产生 G6P，从而完成循环，并使糖基水解酶返回到其初始 NAD/Mn(2+) 配体活性状态。 蔗糖是聚糖合成的前体，有利于口腔病原体（例如变形链球菌）附着在牙齿表面。随后这种二糖和其他二糖发酵（生成乳酸），通过促进牙釉质脱矿质引发龋齿。人们相信微生物无法代谢蔗糖的五种异构体，这表明了这些“甜味”的潜力。非致龋化合物作为膳食蔗糖的替代品，以对抗龋齿的病因。然而，微生物生物化学和遗传学部分进行的创新研究表明，包括梭杆菌、克雷伯氏菌、芽孢杆菌和梭状芽胞杆菌在内的几种细菌物种会迅速异化这些异构体（通常称为：海藻糖、松二糖、麦芽酮糖、明串珠菌糖和帕拉金糖）。独特的转运蛋白和 NAD+/Mn(2+) 依赖性磷酸-α-糖基水解酶参与蔗糖异构体的细菌代谢。相关基因已被克隆、测序并表达蛋白质以进行生化表征。包括变形链球菌在内的口腔链球菌中缺乏这些基因，解释了这些物种未能发酵异构体化合物。鉴于遗传信息 (DNA) 跨物种转移的潜力，我们的研究表明，在广泛使用帕拉金糖和亮氨酸作为膳食蔗糖的替代品时应谨慎行事。重要的是，蔗糖磷酸化衍生物及其异构体的溶液态构象的确定，以及我们对家族 4 水解酶的结构分析，可能允许“基于蔗糖”的合理设计。选择性靶向口腔病原体的抑制剂。