Control Of Sugar Transport & Metabolism In Oral Bacteria

本实验室以前的研究涉及微生物对糖的运输和代谢机制，导致发现了一个大的，但以前未被认识的糖基水解酶（GH）家族。这些新的酶催化各种各样的磷酸化二糖，包括麦芽糖-6？P，纤维二糖-6？P，最显著的是，蔗糖的五种磷酸化异构体。然而，将这些水解酶（命名为家族GH4）与包含糖基水解酶超家族的> 90个家族中的所有其它水解酶区分开的特征是它们对NAD+、二价金属离子和还原条件的强制性要求。这些独特的辅因子是否具有催化或结构能力，直到最近才被发现。然而，在过去的一年中，我们与国际研究人员的合作已经提供了来自枯草芽孢杆菌的磷酸-α-葡糖苷酶（GlvA）与其配体的复合物的晶体结构，分辨率为2.05埃。结合机理研究和溶剂同位素交换，活性位点结构的分析表明糖苷水解需要NAD（H）和Mn（2+）离子参与的新机制。所提出的四步反应涉及C3处的氢化物萃取和NAD+介导的3-OH基团氧化成酮。该氧化步骤导致C2质子的酸化，并通过酶促碱促进去质子化。此后，酸催化的反应导致糖苷氧的消除，并伴随形成1，2-不饱和中间体.这种迈克尔样受体经历水的碱催化攻击，产生葡萄糖6-磷酸（G6 P）的3-酮形式。最后，这种酮中间体减少？在船上吗在一个实施方案中，糖基水解酶与NADH反应以产生G6 P，从而完成循环，并使糖基水解酶返回其初始NAD/Mn（2+）-配体的活性状态。 与阿贡国家实验室、西北大学和约克大学的研究人员进行的相关研究导致了第一次结晶，并确定了来自家族4的磷酸-β-葡萄糖苷酶（BglT）的结构。通过单波长反常色散（SAD）方法在2.85埃分辨率下测定蛋白质的天然结构。在2.55埃分辨率下测定酶与NAD+/Mn 2+和Glc 6P的复合物。BglT与GlvA的活性中心结构的比较，揭示了一个惊人的程度的建筑相似性，在光的动力学同位素效应，允许假设一个共同的反应机制的α-和β-glycoproteinases。这些结构比较表明，简单的空间因素，包括微妙的修改蛋白质折叠，足以调节特异性的共同催化框架。 蔗糖是聚糖合成的前体，其促进口腔病原体的附着，变形链球菌附着在牙齿表面。这种二糖和其他二糖的后续发酵（转化为乳酸）通过促进牙釉质的脱矿作用引发龋齿。相信微生物是无法代谢的五种异构体的蔗糖，表明这些潜力？甜吗？非致龋化合物作为膳食蔗糖的替代物，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（即：海藻酮糖、松二糖、麦芽酮糖、明串珠菌二糖和帕拉金糖）被几种细菌物种（包括梭菌属、克雷伯氏菌属、芽孢杆菌属和梭菌属）迅速异化。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，并表达蛋白质的生化特征。这些基因在口腔链球菌中的缺失，包括链球菌。变形菌，解释了这些物种不能发酵异构化合物。 Berhard Erni博士（伯尔尼大学）目前的研究促进了相关运输和磷酸水解基因转移到大肠杆菌中。在存在葡萄糖：磷酸转移酶系统（EIIAglc）的活性组分的情况下，该生物体还获得以蔗糖异构体和相关的α-连接的葡糖苷为代价生长的能力。

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. Related studies with researchers at Argonne National Lab, Northwestern University and York University have resulted in the first crystallization, and determination of structure of a phospho-beta-glucosidase (BglT) from Family 4. The native structure of the protein was determined by single-wavelength anomalous dispersion (SAD) methods at 2.85 Angstrom resolution. Complexes of the enzyme with NAD+/Mn2+ and Glc6P were determined at 2.55 Angstrom resolution. Comparison of the active-centre structure of BglT with GlvA, reveals a striking degree of architectural similarity, that in light of kinetic isotope effects, allows postulation of a common reaction mechanism for both alpha- and beta-glycosidases. These structural comparisons suggest that simple steric factors, including subtle modifications to protein fold, are sufficient to modulate specificity on a common catalytic framework. Sucrose is the precursor for glycan synthesis that facilitates attachment of oral pathogens e.g., 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, namely : 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 characterizatioon. The absence of these genes in oral streptococci including S. mutans, explains the failure of these species to ferment the isomeric compounds. Current studies with Dr. Berhard Erni (University of Berne) have facilitated the transfer of the relevant transport and phospho-hydrolysis genes to Escherichia coli. In the presence of an active component of the glucose:phosphotransferase system (EIIAglc), this organism also aquires the capacity to grow at the expense of sucrose isomers and related alpha-linked glucosides.