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.
Highlights
Phosphoryl group transfer is a widely used signalling transfer mechanism in living organisms, ranging from bacteria to animal cells
The haloalkanoate dehalogenase (HAD) superfamily is one of the largest and most ubiquitous enzyme families identified to date ( 48 000 sequences reported; http://pfam.sanger.ac.uk/clan?acc=CL0137) and it is Abbreviations HAD, haloalkanoate dehalogenase; isopropyl thio-b-d-galactoside (IPTG), isopropyl thio-b-D-galactoside; pNPP, 4-nitrophenyl phosphate; pNPPase, p-nitrophenyl phosphatase
We report the overproduction, purification and characterization of the AraL enzyme from B. subtilis
Summary
Phosphoryl group transfer is a widely used signalling transfer mechanism in living organisms, ranging from bacteria to animal cells. The resulting plasmids, pLG5 and pLG12 (Fig. 1), bearing the different versions of the recombinant AraL, respectively, under the control of a T7 promoter, were introduced into E. coli BL21(DE3) pLysS (Table 1) for the over-expression of the recombinant proteins.
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