Dataset Information

Characterization and regulation of a bacterial sugar phosphatase of the haloalkanoate dehalogenase superfamily, AraL, from Bacillus subtilis.

ABSTRACT: AraL from Bacillus subtilis is a member of the ubiquitous haloalkanoate dehalogenase superfamily. The araL gene has been cloned, over-expressed in Escherichia coli and its product purified to homogeneity. The enzyme displays phosphatase activity, which is optimal at neutral pH (7.0) and 65 °C. Substrate screening and kinetic analysis showed AraL to have low specificity and catalytic activity towards several sugar phosphates, which are metabolic intermediates of the glycolytic and pentose phosphate pathways. On the basis of substrate specificity and gene context within the arabinose metabolic operon, a putative physiological role of AraL in the detoxification of accidental accumulation of phosphorylated metabolites has been proposed. The ability of AraL to catabolize several related secondary metabolites requires regulation at the genetic level. In the present study, using site-directed mutagenesis, we show that the production of AraL is regulated by a structure in the translation initiation region of the mRNA, which most probably blocks access to the ribosome-binding site, preventing protein synthesis. Members of haloalkanoate dehalogenase subfamily IIA and IIB are characterized by a broad-range and overlapping specificity anticipating the need for regulation at the genetic level. We provide evidence for the existence of a genetic regulatory mechanism controlling the production of AraL.

SUBMITTER: Godinho LM

PROVIDER: S-EPMC3207120 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Characterization and regulation of a bacterial sugar phosphatase of the haloalkanoate dehalogenase superfamily, AraL, from Bacillus subtilis.

Godinho Lia M LM de Sá-Nogueira Isabel I

The FEBS journal 20110602 14

AraL from Bacillus subtilis is a member of the ubiquitous haloalkanoate dehalogenase superfamily. The araL gene has been cloned, over-expressed in Escherichia coli and its product purified to homogeneity. The enzyme displays phosphatase activity, which is optimal at neutral pH (7.0) and 65 °C. Substrate screening and kinetic analysis showed AraL to have low specificity and catalytic activity towards several sugar phosphates, which are metabolic intermediates of the glycolytic and pentose phospha ...[more]

PMID: 21575135

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Project description:5'-nucleotidases (EC 3.1.3.5) catalyze the hydrolytic dephosphorylation of 5'-ribonucleotides and 5'-deoxyribonucleotides as well as complex nucleotides, such as uridine 5'-diphosphoglucose (UDP-glucose), nicotinamide adenine dinucleotide and flavin adenine dinucleotide, to their corresponding nucleosides plus phosphate. These enzymes have been found in diverse species in intracellular and membrane-bound, surface-localized forms. Soluble forms of 5'-nucleotidases belong to the ubiquitous haloacid dehalogenase superfamily (HADSF) and have been shown to be involved in the regulation of nucleotide, nucleoside and nicotinamide adenine dinucleotide (NAD+) pools. Despite the important role of 5'-nucleotidases in cellular metabolism, only a few of these enzymes have been characterized in the Gram-positive bacterium Bacillus subtilis, the workhorse industrial microorganism included in the Food and Drug Administration's GRAS (generally regarded as safe) list. In the present study, we report the identification of a novel 5'-nucleotidase gene from B. subtilis, yutF, which comprises 771 bp encoding a 256-amino-acid protein belonging to the IIA subfamily of the HADSF. The gene product is responsible for the major p-nitrophenyl phosphatase activity in B. subtilis. The yutF gene was overexpressed in Escherichia coli, and its product fused to a polyhistidine tag was purified and biochemically characterized as a soluble 5'-nucleotidase with broad substrate specificity. The recombinant YutF protein was found to hydrolyze various purine and pyrimidine 5'-nucleotides, showing preference for 5'-nucleoside monophosphates and, specifically, 5'-XMP. Recombinant YutF also exhibited phosphohydrolase activity toward nucleotide precursors, ribose-5-phosphate and 5-phosphoribosyl-1-pyrophosphate. Determination of the kinetic parameters of the enzyme revealed a low substrate specificity (Km values in the mM concentration range) and modest catalytic efficiencies with respect to substrates. An initial study of the regulation of yutF expression showed that the yutF gene is a component of the yutDEF transcription unit and that YutF overproduction positively influences yutDEF expression.

Dataset Information

Characterization and regulation of a bacterial sugar phosphatase of the haloalkanoate dehalogenase superfamily, AraL, from Bacillus subtilis.

Publications

Characterization and regulation of a bacterial sugar phosphatase of the haloalkanoate dehalogenase superfamily, AraL, from Bacillus subtilis.

Similar Datasets

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