Sugar Transport /Metabolism In Lactic Acid & oral Bacter

Clostridium acetobutylicum 824基因组包含两个编码NAD、Mn（2+）离子和二硫苏糖醇依赖性磷酸- α -葡萄糖苷酶的基因，可归属于糖基水解酶超家族的第4家族。这两个基因分别被命名为malh（麦芽糖6-磷酸水解酶）和pagl（磷酸- α -葡萄糖苷酶），它们位于不同的操作子中，也编码磷酸烯醇丙酮酸依赖的糖磷酸转移酶系统（PEP-PTS）的蛋白质。C. acetobutylicum生长在多种α -连接的糖苷上，包括麦芽糖、甲基α - d -糖苷和蔗糖的五种异构体。在必要的辅助因子存在的情况下，这些细胞的提取物很容易水解显色底物对硝基苯基- α - d -葡萄糖吡喃苷6-磷酸（pNPalphaGlc6P），但水解是否反映了malH或pagL基因编码的酶的表达，通过分光光度分析或聚丙烯酰胺凝胶电泳无法辨别。要解决这个问题，需要克隆malH和pagL基因，然后分别对麦芽糖6-磷酸水解酶（malH）和磷酸α -葡萄糖苷酶（pagL）进行高表达、纯化和鉴定。MalH和PagL都是四聚体，由相似的？大小（~50 kDA）的亚基，它们具有50%的残基同一性。这两个蛋白与来自mortiferum梭杆菌的抗磷酸- α -葡萄糖苷酶多克隆兔抗体发生交叉反应。虽然纯化的MalH和PagL裂解pNPaGlc6P的效率相当，但只有MalH催化水解通过PEP-PTS形成的6-磷酸二糖。通过电喷雾电离/质谱分析，C. acetobutylicum在所有α -葡糖苷的生长过程中都诱导了MalH，而PagL仅在先前在麦芽糖和甲基- α - d -葡糖苷上生长的生物体的提取物中检测到。我们的研究提供了证据：(i)由mal操纵子编码的蛋白质促进了C. acetobutylicum 824对麦芽糖和相关o - α -连接的糖苷的运输和异化；（ii）磷酸- α -葡萄糖苷酶活性需要一个保守的半胱氨酸残基，（iii）二硫苏糖醇可能通过防止亚基间和亚基内二硫键的形成而有助于酶的稳定性。

The genome of Clostridium acetobutylicum 824 contains two genes encoding NAD, Mn(2+) ion, and dithiothreitol-dependent phospho-alpha-glucosidases that can be assigned to Family 4 of the glycosylhydrolase superfamily. The two genes, designated malh (maltose 6-phosphate hydrolase) and pagl (phospho-alpha-glucosidase) respectively, reside in separate operons that also encode proteins of the phosphoenolpyruvate-dependent:sugar phosphotransferase system (PEP-PTS). C. acetobutylicum grows on a variety of alpha-linked glucosides, including maltose, methyl-alpha-D-glucoside and the five isomers of sucrose. In the presence of the requisite cofactors, extracts of these cells readily hydrolyzed the chromogenic substrate p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNPalphaGlc6P), but whether hydrolysis reflected expression of enzymes encoded by the malH or pagL genes was not discernable by spectro- photometric analysis or polyacryl- amide gel electrophoresis. Resolution of this question required the cloning of the malH and pagL genes, and subsequent high-expression, purification, and characterization of maltose 6-phosphate hydrolase (MalH) and phospho-alpha-glucosidase (PagL), respectively. Both MalH and PagL are tetramers comprised of similar?sized (~50 kDA) subunits, and they exhibit 50% residue identity. The two proteins cross-react with polyclonal rabbit antibody against phospho-alpha-glucosidase from Fusobacterium mortiferum. Although purified MalH and PagL cleaved pNPaGlc6P with comparable efficiency, only MalH catalyzed the hydrolysis of disaccharide 6-phosphates formed via the PEP-PTS. Analysis of the proteome of C. acetobutylicum by electrospray ionization/mass spectrometry revealed induction of MalH during growth on all alpha-glucosides tested, whereas PagL was detected only in extracts prepared from organisms grown previously on maltose and methyl-alpha-D-glucoside. Our studies provide evidence: (i) that proteins encoded by the mal operon facilitate the transport and dissimilation of maltose and related O-alpha-linked glucosides, by C. acetobutylicum 824; (ii) that a conserved cysteine residue is required for phospho-alpha-glucosidase activity, and (iii) that dithiothreitol may contribute to enzyme stability by preventing the formation of inter- and intra -subunit disulfide bonds.