L. M. Godinho and I. de Sa´-Nogueira AraL sugar phosphatase from B. subtilis pLG11 pLG12/pLG13 M pLG5 rbs M R I M A S H D T P V S P A G I L I D ATCGAAAACACGGAGCAAATGCGTATTATGGCCAGTCATGATACGCCTGTGTCACCGGCTGGCATTCTGATTGAC A A A 2947.9 kb araA araB araD araL araM araN araP araQ abfA araA araB araD araM araN araP araQ abfA WT IQB832 Fig. 1. Schematic representation of the araL genomic context in B. subtilis. White arrows pointing in the direction of transcription represent the genes in the arabinose operon, araABDLMNPQ-abfA. The araL gene is highlighted in grey and the promoter of the transcriptional unit is depicted by a black arrow. Depicted below the araABDLMNPQ-abfA is the in-frame deletion generated by allelic replacement DaraL. Above is displayed the coding sequence of the 5¢-end of the araL gene. The putative ribosome-binding site, rbs, is underlined. The 5¢-end of the araL gene present in the different constructs pLG5, pLG11, pLG12 and pLG13, is indicated by an arrow above the sequence. Mutations introduced in the construction of pLG11, pLG13 and pLG26 are indicated below the DNA sequence and the corresponding modiﬁcation in the primary sequence of AraL is depicted above. Homologs of the B. subtilis AraL protein are found in different species of Bacteria and Archea, and genes encoding proteins with more than 50% amino acid identity to AraL are present in Bacillus and Geobacillus species, clustered together with genes involved in arabi- nose catabolism. An alignment of the primary sequence of AraL with other members of the NagD family from different organisms, namely NagD from E. coli (27% identity), the p-nitrophenyl phosphatases (pNPPases) from S. cerevisiae (24% identity), Sz. pom- be (30% identity) and Plasmodium falciparum (31% identity), highlights the similarities and differences (Fig. 2). AraL displays the conserved key catalytic resi- dues that unify HAD members: the Asp at position 9 (motif I) together with Asp 218 (motif IV) binds the cofactor Mg2+, and Ser 52 (motif II) together with Lys 193 (motif III) binds the phosphoryl group (Fig. 2). The cap domain is responsible for substrate binding ⁄ speciﬁcity; thus, the uniqueness or similarity of the amino acid sequence in this domain may deter- mine enzyme speciﬁcity or the lack thereof [10,13,20]. Similar to the other members of the NagD family, AraL shares two Asp residues in the cap domain (Fig. 2). To date, the number of characterized mem- bers of this family is scarce. In the present study, we show that AraL possesses activity towards different sugar phosphates. The NagD enzyme was observed to have a nucleotide phosphohydrolase activity coupled with a sugar phosphohydrolase activity [13]. The P. falciparum enzyme displayed nucleotide and sugar phosphatase activity together with an ability to dephosphorylate the vitamin B1 precursor thiamine monophosphate [21]. The yeast’s enzymes are p-nitro- phenyl phosphatases; however, natural substrates were not found [16,17]. The majority of the enzymes dis- played in this alignment show activity to overlapping sugar phosphates [13,21] and it is tempting to speculate that this is related to similarities in the cap domain. On the other hand, the variability and dissimilarity observed in this region may determine the preference for certain substrates (Fig. 2). Overproduction and puriﬁcation of recombinant AraL Full-length araL coding regions, starting at both the ﬁrst and second putative initiation ATG codons, were separately cloned in the expression vector pET30a(+) (Table 1), which allows the insertion of a His6-tag at the C-terminus. 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 recom- binant proteins. The cells were grown in the presence and absence of the inducer isopropyl thio-b-d-galacto- side (IPTG), and soluble and insoluble fractions were prepared as described in the Experimental procedures and analyzed by SDS ⁄ PAGE. In both cases, the pro- duction of AraL was not detected, although different methodologies for over-expression have been used (see below). On the basis on the alignment of the primary sequence of AraL and NagD, we constructed a truncated version FEBS Journal 278 (2011) 2511–2524 ª 2011 The Authors Journal compilation ª 2011 FEBS 2513