Regulation Of Sugar Transport And Metabolism In Lactic A

本实验室先前对微生物运输和代谢糖的机制进行了研究，发现了一个大的，但以前未被认识的糖基水解酶（GH）家族。这些新型酶催化多种磷酸化双糖的裂解，包括麦芽糖-6？P, cellobiose-6 ?P和最显著的是蔗糖的五个磷酸化异构体。然而，将这些水解酶（指定为GH4家族）与构成糖基水解酶超家族的bbbb90家族中的所有其他水解酶区分开来的特征是它们对NAD+、二价金属离子和活性还原条件的必需要求。直到最近，这些独特的辅因子是否在催化或结构能力中起作用还是未知的。然而，在过去的一年里，我们与国际研究人员合作，提供了枯草芽孢杆菌磷酸- 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，表明糖苷水解的新机制需要NAD(H)和Mn（2+）离子的参与。提出的四步反应包括在C3处的氢化物萃取，以及NAD+介导的3-OH基团氧化成酮。这个氧化步骤引起C2质子的酸化，并促进酶碱的去质子化。此后，酸催化反应导致糖苷氧的消除，并随之形成1,2 -不饱和中间体。这种类米迦勒受体受到水的碱催化攻击，生成3-酮形式的葡萄糖6-磷酸（G6P）。最后，这种酮-中间体被板上的？NADH生成G6P，从而完成循环，并使糖基水解酶恢复到初始的NAD/Mn（2+）配体活性状态。

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.