AraL sugar phosphatase from B. subtilis L. M. Godinho and I. de Sa´-Nogueira highly represented in individual cells. The family was named after the archetypal enzyme, haloacid dehalo- genase, which was the ﬁrst family member to be struc- turally characterized [4,5]. However, it comprises a wide range of HAD-like hydrolases, such as phospha- tases ( 79%) and ATPases (20%), the majority of which are involved in phosphoryl group transfer to an active site aspartate residue [6–8]. HAD phosphatases are involved in variety of essential biological functions, such as primary and secondary metabolism, mainte- nance of metabolic pools, housekeeping functions and nutrient uptake [8]. The highly conserved structural core of the HAD enzymes consists of a a-b domain that adopts the topology typical of the Rossmann a ⁄ b folds, housing the catalytic site, and is distinguished from all other Rossmanoid folds by two unique struc- tural motifs: an almost complete a-helical turn, named the ‘squiggle’, and a b-hairpin turn, termed the ‘ﬂap’ [6,8,9]. The HAD superfamily can be divided into three generic subfamilies based on the existence and location of a cap domain involved in substrate recognition. Subfamily I possesses a small a-helical bundle cap between motifs I and II; subfamily II displays a cap between the second and third motifs; and subfamily III members present no cap domain [10]. Subfamily IIA, based on the topology of the cap domain, can be further divided into two subclasses: subclass IIA and subclass IIB [10]. Presently, 2000 sequences are assigned to HAD subfamily IIA, which covers humans and other eukary- otes, as well as Gram-positive and Gram-negative bacte- ria (http://www.ebi.ac.uk/interpro/IEntry?ac=IPR006357). The Escherichia coli NagD [11] and the Bacillus subtilis putative product AraL [12] typify this subfamily. NagD is a nucleotide phosphatase, encoded by the nagD gene, which is part of the N-acetylglucosamine operon (nag- BACD). The puriﬁed enzyme hydrolyzes a number of phosphate containing substrates, and it has a high spec- iﬁcity for nucleotide monophosphates and, in particu- lar, UMP and GMP. The structure of NagD has been determined and the occurrence of NagD in the context of the nagBACD operon indicated its involvement in the recycling of cell wall metabolites [13]. Although this subfamily is widely distributed, only few members have been characterized. In the present study, we report the overproduction, puriﬁcation and characterization of the AraL enzyme from B. subtilis. AraL is shown to be a phosphatase displaying activity towards different sugar phosphate substrates. Furthermore, we provide evidence that, in both E. coli and B. subtilis, production of AraL is regulated by the formation of an mRNA secondary structure, which sequesters the ribosome-binding site and consequently prevents translation. AraL is the ﬁrst sugar phosphatase belonging to the family of NagD- like phosphatases to be characterized at the level of gene regulation. Results and Discussion The araL gene in the context of the B. subtilis genome and in silico analysis of AraL The araL gene is the fourth cistron of the transcrip- tional unit araABDLMNPQ-abfA [12]. This operon is mainly regulated at the transcriptional level by induc- tion in the presence of arabinose and repression by the regulator AraR [14,15]. To date, araL is the only un- characterized ORF present in the operon (Fig. 1). The putative product of araL displays some similarities to p-nitrophenyl phosphate-speciﬁc phosphatases from the yeasts Saccharomyces cerevisiae and Schizosacchar- omyces pombe [16,17] and other phosphatases from the HAD superfamily, namely the NagD protein from E. coli [13]. Although the yeast enzymes were identiﬁed as phosphatases, no biologically relevant substrate could be determined, and both enzymes appeared to be dispensable for vegetative growth and sporulation. The puriﬁed NagD hydrolyzes a number of nucleotide and sugar phosphates. The araL gene contains two in-frame ATG codons in close proximity (within 6 bp; Fig. 1). The sequence reported by Sa´ -Nogueira et al. [12] assumed that the second ATG, positioned further downstream (Fig. 1), was the putative start codon for the araL gene because its distance relative to the ribosome-binding site is more similar to the mean distance (5–11 bp) observed in Bacillus [18]. However, in numerous databases, the upstream ATG is considered as the initiation codon [19]. Assuming that the second ATG is correct, the araL gene encodes a protein of 269 amino acids with a molecular mass of 28.9 kDa. HAD family members are identiﬁed in amino acid alignments by four active site loops that form the mechanistic gear for phosphoryl transfer [8]. The key residues are an aspartate in motif I (D), a serine or threonine motif II (S ⁄ T), an arginine or lysine motif III (R ⁄ K) and an aspartate or glutamate motif IV (D ⁄ E). The NagD family members display a unique a ⁄ b cap domain that is involved in substrate recogni- tion, located between motifs II and III [6]. This family is universally spread; however, only a few members have been characterized, such as NagD from E. coli [6,11]. NagD members are divided into different sub- families, such as the AraL subfamily [6], although all proteins present a GDxxxxD motif IV (Fig. 2). 2512 FEBS Journal 278 (2011) 2511–2524 ª 2011 The Authors Journal compilation ª 2011 FEBS