Characterization and regulation of a bacterial sugar phosphatase of the haloalkanoate dehalogenase superfamily, AraL, from Bacillus subtilis Lia M. Godinho and Isabel de Sa´ -Nogueira Centro de Recursos Microbiolo´ gicos, Departamento de Ci eˆncias da Vida, Faculdade de Cieˆ ncias e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, Caparica, Portugal Keywords AraL; Bacillus subtilis; gene regulation; HAD superfamily (IIA); sugar phosphatase Correspondence I. de Sa´-Nogueira, Departamento de Ci eˆncias da Vida, Faculdade de Cieˆ ncias e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal Fax: +351 21 2948530 Tel: +351 21 2947852 E-mail: isn@fct.unl.pt Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/ onlineopen#OnlineOpen_Terms (Received 2 October 2010, revised 1 April 2011, accepted 10 May 2011) doi:10.1111/j.1742-4658.2011.08177.x 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 path- ways. 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 sec- ondary 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 sub- family IIA and IIB are characterized by a broad-range and overlapping speciﬁcity anticipating the need for regulation at the genetic level. We pro- vide evidence for the existence of a genetic regulatory mechanism control- ling the production of AraL. Introduction Phosphoryl group transfer is a widely used signalling transfer mechanism in living organisms, ranging from bacteria to animal cells. Phosphate transfer mecha- nisms often comprise a part of the strategies used to respond to different external and internal stimuli, and protein degradation [1]. Phosphoryl-transfer reactions, catalysed by phosphatases, remove phosphoryl groups from macromolecules and metabolites [2]. It is esti- mated that 35–40% of the bacterial metabolome is composed of phosphorylated metabolites [3]. The majority of cellular enzymes responsible for phos- phoryl transfer belong to a rather small set of super- families that are all evolutionary distinct, with different structural topologies, although they are almost exclusively restricted to phosphoryl group transfer. The haloalkanoate dehalogenase (HAD) superfamily is one of the largest and most ubiquitous enzyme fami- lies identiﬁed to date ( 48 000 sequences reported; http://pfam.sanger.ac.uk/clan?acc=CL0137) and it is Abbreviations HAD, haloalkanoate dehalogenase; IPTG, isopropyl thio-b-D-galactoside; pNPP, 4-nitrophenyl phosphate; pNPPase, p-nitrophenyl phosphatase. FEBS Journal 278 (2011) 2511–2524 ª 2011 The Authors Journal compilation ª 2011 FEBS 2511