Sugar Transport /Metabolism In Lactic Acid & oral Bacter

乙酰丁基梭菌824的基因组包含两个编码NAD、Mn(2+)和二硫苏糖醇依赖的磷酸-α-葡萄糖苷酶的基因，它们属于糖基水解酶超家族中的第4家族。这两个基因分别命名为MALH(麦芽糖6-磷酸水解酶)和PAGE(磷酸-α-葡萄糖苷酶)，分别位于不同的操纵子中，这些操纵子也编码磷酸烯醇式丙酮酸依赖的糖磷酸转移酶系统(PEP-PTS)的蛋白质。乙酰丁酸单胞菌生长在多种α-连接的糖苷上，包括麦芽糖、甲基-α-D-葡萄糖苷和蔗糖的五种异构体。在所需辅因子的存在下，这些细胞的提取物很容易对显色底物p-硝基苯基-α-D-吡喃葡萄糖苷6-磷酸(PNPalphaGlc6P)进行水解，但通过分光光度分析或聚丙烯酰胺凝胶电泳法无法区分水解物是否反映了由MalH或PagL基因编码的酶的表达。这个问题的解决需要克隆MALH和PagL基因，随后分别高效表达、纯化和鉴定麦芽糖6-磷酸水解酶(MALH)和磷酸-α-葡萄糖苷酶(PagL)。MalH和PagL都是由大小相似(约50kda)的亚基组成的四聚体，它们显示出50%的残基同源性。这两种蛋白与抗Mortiferum磷酸-α-葡萄糖苷酶的多克隆兔抗体发生交叉反应。虽然纯化的MalH和PagL以相似的效率切割pNPaGlc6P，但只有Malh催化通过PEP-PTS形成的6-磷酸二糖的水解。电喷雾电离/质谱仪对乙酰丁酸杆菌蛋白质组的分析表明，在所有受试的α-葡萄糖苷生长过程中都诱导了MalH的产生，而PagL仅在先前在麦芽糖和甲基-α-D-葡萄糖苷上生长的微生物的提取物中检测到。我们的研究提供了证据：(I)由麦芽糖操纵子编码的蛋白质促进了麦芽糖和相关的O-α连接的葡萄糖苷的运输和异化，由乙酰丁酸杆菌824；(Ii)保守的半胱氨酸残基是磷酸-α-葡萄糖苷酶活性所必需的，以及(Iii)二硫苏糖醇可能通过阻止亚单位间和亚基内二硫键的形成来促进酶的稳定性。

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.