Regulation Of Sugar Transport And Metabolism In Oral Bacteria

粪肠球菌的麦芽糖分解 与枯草芽孢杆菌相似，粪肠球菌通过磷酸烯醇丙酮酸（PEP）：麦芽糖磷酸转移酶系统（PTS）转运和磷酸化麦芽糖。麦芽糖特异性PTS通透酶由基因malT编码。然而，E.粪肠球菌缺乏编码6-磷酸-α-葡糖苷酶的malA基因，该基因在B.相反，编码麦芽糖磷酸化酶（MalP）、磷酸葡萄糖变位酶和变旋酶的操纵子从malT的上游开始。MalP被认为可以将麦芽糖-6-P分解为葡萄糖-1-P和葡萄糖-6-P。然而，纯化的MalP磷酸化麦芽糖，但不磷酸化麦芽糖-6-P。我们发现malT下游的基因编码一种新型酶（MapP），该酶可以将PTS形成的麦芽糖-6-P去磷酸化。所得的细胞内麦芽糖被MalP水解成葡萄糖和葡萄糖-1-P。通过麦芽糖糊精ABC转运蛋白缓慢摄取麦芽糖仅允许mapP而不是malP突变体生长不良。在B中合成MapP。积累麦芽糖-6-P的枯草杆菌突变株在麦芽糖上恢复生长。MapP催化细胞内麦芽糖-6-P的去磷酸化，并且所产生的麦芽糖被B转化。因此，MapP将PTS介导的麦芽糖摄取与麦芽糖磷酸化酶催化的代谢联系起来。用多种磷酸化底物进行的去磷酸化测定显示，MapP优选使含有O-α-糖基连接的二糖去磷酸化。这些发现已发表在《Molecular Microbiology》上。 肺炎链球菌TIGR 4磷酸-β-葡萄糖苷酶（BGLA-2）的结构与功能 肺炎链球菌是急性肺炎、中耳炎、脑膜炎和败血症的主要病原体，其每年导致全世界数百万人死亡。在人类宿主中，S.肺炎遇到多种糖缀合物，包括粘蛋白、宿主防御分子和上皮细胞上的表面暴露聚糖。与其他病原微生物一样，S。肺炎产生多种分泌的或表面相关的糖苷酶，其功能包括宿主糖缀合物的修饰和水解。基因组测序，结合新的毒力因子的探索，表明大量的糖苷酶是必要的最大毒力的S。肺炎。BglA-2由S. pneumoniae TIGR4.在高表达载体中克隆该基因后，将BglA-2（471个残基，MW 54，361）纯化至电泳均一。磷酸-β-葡糖苷酶（BglA-2）的天然底物包括：纤维二糖-6-磷酸、龙胆二糖-6P、熊果苷-6P、水杨苷-6P和相关的0-β-连接的二糖磷酸。 使用这些新的化合物允许底物特异性和动力学分析进行。来自糖苷水解酶家族1（GH-1）的6-磷酸-β-葡糖苷酶BglA-2（EC 3.2.1.86）催化β-1，4-连接的纤维二糖-6-磷酸的水解以产生葡萄糖和葡萄糖-6-磷酸（G6 P）。两种反应产物都通过产生能量的糖酵解途径进一步代谢。在这项研究中，我们提出了第一晶体结构的载脂蛋白和复合物形式的BglA-2与硫代纤维二糖-6P（纤维二糖-6P的非代谢类似物）在2.0和2.4埃分辨率，分别。与其它GH-1酶类似，S.肺炎克雷伯氏菌采用典型的（β/α）8 TIM-桶，其中活性位点位于β-桶的凸面的中心。结合从定点突变蛋白获得的酶数据，结构分析表明BglA-2亚位点+1处的三个芳香族残基：Tyr 126、Tyr 303和Trp 338决定了相对于（1，4）-连接的6-磷酸-β-葡糖苷底物的底物特异性。此外，三个额外的残基：丝氨酸424，赖氨酸430和酪氨酸432的BglA-2，被发现发挥重要作用的水解选择性对磷酸化，而不是非磷酸化的化合物。比较结构分析表明，亚位点-1处的色氨酸与甲硫氨酸/丙氨酸残基可能导致结构相似的酶6-磷酸-β-半乳糖苷酶和6-磷酸-β-葡糖苷酶之间的催化和底物差异，归属于糖苷水解酶超家族的GH-1家族。我们的研究结果发表在《生物化学杂志》上。 B的LPLD。枯草杆菌是一种α-半乳糖醛酸苷酶，属于糖苷水解酶家族4。 在早期的系统发育分析201 GH4酶，我们注意到一组未知的催化活性的基序CHEV的蛋白质。2008年报道了这些蛋白质之一，来自枯草芽孢杆菌菌株168的LplD的结构，但未测定酶活性。在过去的一年中，与美国和加拿大的研究人员合作，我们已经证明含有CHEV基序的蛋白质是α-半乳糖醛酸酶，其天然底物是α-1，4-二-半乳糖醛酸（GalUA 2）。调查结果发表在欧洲统计局通讯上。

MALTOSE DISSIMILATION IN ENTEROCOCCUS FAECALIS. Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP): maltose phospho- transferase system (PTS). The maltose - specific PTS permease is encoded by the gene malT. However, E. faecalis lacks a malA gene encoding a 6-phospho-alpha-glucosidase which in B. subtilis hydrolyses maltose-6-P into glucose and glucose-6-P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose-6-P into glucose-1-P and glucose-6-P. However, purified MalP phosphorolyses maltose but not maltose-6-P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose-6-P formed by the PTS. The resulting intracellular maltose is hydrolyzed by MalP into glucose and glucose-1-P. Slow uptake of maltose via a maltodextrin ABC transporter allows poor growth only for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose-6-P restored growth on maltose. MapP catalyzes the dephosphorylation of intracellular maltose-6-P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose-1-P. MapP therefore connects PTS-mediated maltose uptake to maltose phosphorylase-catalyzed metabolism. Dephosphorylation assays with a wide variety of phosphorylated substrates revealed that MapP preferably dephosphorylates disaccharides containing an O-alpha-glycosyl linkage. These findings have been published in Molecular Microbiology. STRUCTURE AND FUNCTION OF PHOSPHO-BETA-GLUCOSIDASE (BGLA-2) FROM STREPTOCOCCUS PNEUMONIAE TIGR4. Streptococcus pneumonia is the major causative agent of acute pneumonia, otitis media, meningitis, and septicemia, which annually result in the deaths of millions worldwide. In the human host, S. pneumoniae encounters a variety of glyco-conjugates, including mucins, host defense molecules, and surface exposed glycans on epithelial cells. In common with other pathogenic microbes, S. pneumonia produces a variety of secreted or surface-associated glycosidases whose function(s) include the modification and hydrolysis of host glyco-conjugates. Genome sequencing, in combination with exploration of new virulence factors, suggests that a large number of glycosidases are necessary for maximum virulence of S. pneumoniae. BglA-2 is encoded by the gene Sp_0578 in the chromosomal DNA of S. pneumoniae TIGR4. After cloning of the gene in a high expression vector, BglA-2 (471 residues, MW 54,361) was purified to electrophoretic homogeneity. The natural substrates of phospho-beta-glucosidase (BglA-2) include: cellobiose-6-phosphate, gentiobiose-6P, arbutin-6P, salicin-6P and related O-beta-linked disaccharide phosphates. Use of these novel compounds permitted substrate specificity and kinetic analyses to be conducted. The 6-phospho-beta-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of beta-1,4-linked cellobiose -6-phosphate to yield glucose and glucose-6-phosphate (G6P). Both reaction products are further metabolized by the energy - generating glycolytic pathway. In this study, we present the first crystal structures of the apo- and complex-forms of BglA-2 with thiocellobiose-6P (a non-metabolizable analog of cellobiose-6P) at 2.0 and 2.4 Angstrom resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from S. pneumoniae adopts a typical (beta/alpha)8 TIM-barrel, with the active site located at the center of the convex surface of the beta-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues: Tyr126, Tyr303 and Trp338 at subsite +1 of BglA-2, determine substrate specificity with respect to (1,4)-linked 6-phospho-beta-glucoside substrates. Moreover, three additional residues: Ser424, Lys430 and Tyr432 of BglA-2, were found to play important roles in the hydrolytic selectivity towards phosphorylated, rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite -1 may contribute to the catalytic and substrate differences, between the structurally similar enzymes 6-phospho-beta-galactosidase and 6-phospho-beta-glucosidase, assigned to Family GH-1 of the Glycoside Hydrolase superfamily. Our findings have been reported in the Journal of Biological Chemistry. LPLD OF B. SUBTILIS IS AN ALPHA-GALACTURONIDASE ASSIGNED TO GLYCOSIDE HYDROLASE FAMILY 4. In an earlier phylogenetic analysis of 201 GH4 enzymes, we noted a group of proteins of unknown catalytic activity with the motif CHEV. The structure of one of those proteins, LplD from Bacillus subtilis strain 168, was reported in 2008 but the enzymatic activity was not determined. In the past year, in collaboration with investigators in the USA and Canada, we have shown that proteins containing the CHEV motif are alpha-galacturonidase(s) whose natural substrate is alpha-1,4-di-galacturonate (GalUA2). The results obtained from this investigation were published in FEBS Letters.