Dataset Information

Molecular differences between a mutase and a phosphatase: investigations of the activation step in Bacillus cereus phosphopentomutase.

ABSTRACT: Prokaryotic phosphopentomutases (PPMs) are di-Mn(2+) enzymes that catalyze the interconversion of ?-D-ribose 5-phosphate and ?-D-ribose 1-phosphate at an active site located between two independently folded domains. These prokaryotic PPMs belong to the alkaline phosphatase superfamily, but previous studies of Bacillus cereus PPM suggested adaptations of the conserved alkaline phosphatase catalytic cycle. Notably, B. cereus PPM engages substrates when the active site nucleophile, Thr-85, is phosphorylated. Further, the phosphoenzyme is stable throughout purification and crystallization. In contrast, alkaline phosphatase engages substrates when the active site nucleophile is dephosphorylated, and the phosphoenzyme reaction intermediate is only stably trapped in a catalytically compromised enzyme. Studies were undertaken to understand the divergence of these mechanisms. Crystallographic and biochemical investigations of the PPM(T85E) phosphomimetic variant and the neutral corollary PPM(T85Q) determined that the side chain of Lys-240 underwent a change in conformation in response to active site charge, which modestly influenced the affinity for the small molecule activator ?-D-glucose 1,6-bisphosphate. More strikingly, the structure of unphosphorylated B. cereus PPM revealed a dramatic change in the interdomain angle and a new hydrogen bonding interaction between the side chain of Asp-156 and the active site nucleophile, Thr-85. This hydrogen bonding interaction is predicted to align and activate Thr-85 for nucleophilic addition to ?-D-glucose 1,6-bisphosphate, favoring the observed equilibrium phosphorylated state. Indeed, phosphorylation of Thr-85 is severely impaired in the PPM(D156A) variant even under stringent activation conditions. These results permit a proposal for activation of PPM and explain some of the essential features that distinguish between the catalytic cycles of PPM and alkaline phosphatase.

SUBMITTER: Iverson TM

PROVIDER: S-EPMC3302354 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Molecular differences between a mutase and a phosphatase: investigations of the activation step in Bacillus cereus phosphopentomutase.

Iverson T M TM Panosian Timothy D TD Birmingham William R WR Nannemann David P DP Bachmann Brian O BO

Biochemistry 20120221 9

Prokaryotic phosphopentomutases (PPMs) are di-Mn(2+) enzymes that catalyze the interconversion of α-D-ribose 5-phosphate and α-D-ribose 1-phosphate at an active site located between two independently folded domains. These prokaryotic PPMs belong to the alkaline phosphatase superfamily, but previous studies of Bacillus cereus PPM suggested adaptations of the conserved alkaline phosphatase catalytic cycle. Notably, B. cereus PPM engages substrates when the active site nucleophile, Thr-85, is phosp ...[more]

PMID: 22329805

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Project description:BackgroundBacillus cereus is a foodborne pathogen commonly found in nature and food and can cause food spoilage and health issues. Although the prevalence of B. cereus in foods has been reported worldwide, the extent of contamination in edible fungi, which has become increasingly popular as traditional or functional food, is largely unknown. Here we investigated the prevalence, toxin genes' distribution, antibiotic resistance, and genetic diversity of B. cereus isolated from edible fungi in China.ResultsSix hundred and ninety-nine edible fungi samples were collected across China, with 198 (28.3%) samples found to be contaminated by B. cereus, with an average contamination level of 55.4 most probable number (MPN)/g. Two hundred and forty-seven B. cereus strains were isolated from the contaminated samples. Seven enterotoxin genes and one cereulide synthetase gene were detected. The detection frequencies of all enterotoxin genes were ≥ 80%, whereas the positive rate of the cesB gene in B. cereus was 3%. Most isolates were resistant to penicillins, β-lactam/β-lactamase inhibitor combinations, cephems, and ansamycins, but were susceptible to penems, aminoglycosides, macrolides, ketolide, glycopeptides, quinolones, phenylpropanol, tetracyclines, lincosamides, streptogramins, and nitrofurans. Meanwhile, 99.6% of all isolates displayed multiple antimicrobial resistance to three or more classes of antimicrobials. Using genetic diversity analysis, all isolates were defined in 171 sequence types (STs), of which 83 isolates were assigned to 78 new STs.ConclusionsThis study provides large-scale insight into the prevalence and potential risk of B. cereus in edible fungi in China. Approximately one-third of the samples were contaminated with B. cereus, and almost all isolates showed multiple antimicrobial resistance. Detection frequencies of all seven enterotoxin genes were equal to or more than 80%. These new findings may indicate a need for proper pre-/post-processing of edible fungi to eliminate B. cereus, thereby preventing the potential risk to public health.

Dataset Information

Molecular differences between a mutase and a phosphatase: investigations of the activation step in Bacillus cereus phosphopentomutase.

Publications

Molecular differences between a mutase and a phosphatase: investigations of the activation step in Bacillus cereus phosphopentomutase.

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