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Structural insights into the substrate specificity of a 6-phospho-?-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4.

ABSTRACT: The 6-phospho-?-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of ?-1,4-linked cellobiose 6-phosphate (cellobiose-6'P) to yield glucose and glucose 6-phosphate. Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo and complex forms of BglA-2 with thiocellobiose-6'P (a non-metabolizable analog of cellobiose-6'P) at 2.0 and 2.4 ? resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from Streptococcus pneumoniae adopts a typical (?/?)8 TIM-barrel, with the active site located at the center of the convex surface of the ?-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues, Tyr(126), Tyr(303), and Trp(338), at subsite +1 of BglA-2 determine substrate specificity with respect to 1,4-linked 6-phospho-?-glucosides. Moreover, three additional residues, Ser(424), Lys(430), and Tyr(432) of BglA-2, were found to play important roles in the hydrolytic selectivity toward 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 selectivity with respect to structurally similar 6-phospho-?-galactosidases and 6-phospho-?-glucosidases assigned to the GH-1 family.

SUBMITTER: Yu WL

PROVIDER: S-EPMC3663516 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Structural insights into the substrate specificity of a 6-phospho-β-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4.

Yu Wei-Li WL Jiang Yong-Liang YL Pikis Andreas A Cheng Wang W Bai Xiao-Hui XH Ren Yan-Min YM Thompson John J Zhou Cong-Zhao CZ Chen Yuxing Y

The Journal of biological chemistry 20130411 21

The 6-phospho-β-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of β-1,4-linked cellobiose 6-phosphate (cellobiose-6'P) to yield glucose and glucose 6-phosphate. Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo and complex forms of BglA-2 with thiocellobiose-6'P (a non-metabolizable analog of cellobiose-6'P) at 2.0 and 2.4 Å resolution, respective ...[more]

PMID: 23580646

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Project description:Streptococcus pneumoniae (pneumococcus) is a leading human pathogen that can cause serious localized and invasive diseases. Pneumococci can undergo a spontaneous and reversible phase variation that is reflected in colony opacity and which allows the population to adapt to different host environments. Generally, transparent variants are adapted for nasopharyngeal colonization, whereas opaque variants are associated with invasive disease. In recent work, colony phase variation was shown to occur by means of recombination events to generate multiple alleles of the hsdS targeting domain of a DNA methylase complex, which mediates epigenetic changes in gene expression. A panel of isogenic strains were created in the well-studied S. pneumoniae TIGR4 background that are "locked" in the transparent (n = 4) or opaque (n = 2) colony phenotype. The strains had significant differences in colony size which were stable over multiple passages in vitro and in vivo. While there were no significant differences in adherence for the phase-locked mutant strains to immortalized epithelial cells, biofilm formation and viability were reduced for the opaque variants in static assays. Nasopharyngeal colonization was stable for all strains, but the mortality rates differed between them. Transcript profiling by transcriptome sequencing (RNA-seq) analyses revealed that the expression levels of certain virulence factors were increased in a phase-specific manner. As epigenetic regulation of phase variation (often referred to as "phasevarion") is emerging as a common theme for mucosal pathogens, these results serve as a model for future studies of host-pathogen interactions. IMPORTANCE A growing number of bacterial species undergo epigenetic phase variation due to variable expression or specificity of DNA-modifying enzymes. For pneumococci, this phase variation has long been appreciated as being revealed by changes in colony opacity, which are reflected in changes in expression or accessibility of factors on the bacterial surface. Recent work showed that recombination-generated variation in alleles of the HsdS DNA methylase specificity subunit mediated pneumococcal phase variation. We generated phase-locked populations of S. pneumoniae TIGR4 expressing a single nonvariant hsdS allele and observed significant differences in gene expression and virulence. These results highlight the importance of focused pathogenesis studies within specific phase types. Moreover, the generation of single-allele hsdS constructs will greatly facilitate such studies.

Dataset Information

Structural insights into the substrate specificity of a 6-phospho-?-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4.

Publications

Structural insights into the substrate specificity of a 6-phospho-β-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4.

Similar Datasets

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