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 22.214.171.124) 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.
|Yu, Wei-Li; Jiang, Yong-Liang; Pikis, Andreas et al. (2013) Structural insights into the substrate specificity of a 6-phospho-Î²-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4. J Biol Chem 288:14949-58|
|Mokhtari, Abdelhamid; Blancato, VÃctor S; Repizo, Guillermo D et al. (2013) Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose 6'-phosphate phosphatase (MapP). Mol Microbiol 88:234-53|
|Thompson, John; Pikis, Andreas; Rich, Jamie et al. (2013) Î±-Galacturonidase(s): a new class of Family 4 glycoside hydrolases with strict specificity and a unique CHEV active site motif. FEBS Lett 587:799-803|
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