title: 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.
authors.affiliation: Centro de Recursos Microbiológicos, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, Caparica, Portugal.
authors.forename: Lia M
authors.initials: LM
authors.lastname: Godinho
authors.forename: Isabel
authors.initials: I
authors.lastname: de Sá-Nogueira
mesh_terms: D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D019295:Computational Biology; D005103:Expert Systems; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009943:Organophosphorus Compounds / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D050436:Regulatory Elements, Transcriptional; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D000814:Aniline Compounds; D001426:Bacterial Proteins; D007527:Isoenzymes; D009943:Organophosphorus Compounds; D012333:RNA, Messenger; D011994:Recombinant Proteins; C068798:4-aminophenylphosphate; D006867:Hydrolases; C046137:haloalkane dehalogenase