AraL sugar phosphatase from B. subtilis L. M. Godinho and I. de Sa´-Nogueira Table 1. (Continued). Plasmid, strain or oligonucleotide ARA516 ARA549 ARA550 ARA551 ARA552 ARA553 ARA554 Relevant construction, genotype or sequence (5¢- to 3¢) CAC CAC GCT CAT CGA TAA TTT CAC C GGC CAG TCA TGA TAG GCC TGT GTC ACC GGT GAC ACA GGC CTA TCA TGA CTG GCC GCA AAT GCC TAT TAT GGC CAG TCA TGA TAG GCC TGT GTC GAC ACA GGC CTA TCA TGA CTG GCC ATA ATA GGC ATT TGC CGG AGC AAA TGC TTA TTA TGG CCA GTC GAC TGG CCA TAA TAA GCA TTT GCT CCG Source or Reference A kDa 150 100 75 50 37 25 20 15 pET30 P S pLG11 P S B kDa 150 100 75 50 37 25 20 Fig. 3. Overproduction and puriﬁcation of recombinant AraL-His6. (A) Analysis of the soluble (S) and insoluble (P) protein fraction (20 lg of total protein) of induced cultures of E. coli Bl21(DE3) pLysS harboring pET30a (control) and pLG11 (AraL-His6). (B) Analy- sis of different fractions of puriﬁed recombinant AraL eluted with 300 mM imidazole. The proteins were separated by SDS ⁄ PAGE 12.5% gels and stained with Coomassie blue. A white arrowhead indicates AraL-His6. The size (kDa) of the broad-range molecular mass markers (Bio-Rad Laboratories) is indicated. AraL is a sugar phosphatase AraL is a phosphatase displaying activity towards the synthetic substrate pNPP, although there is no evi- dence that pNPPase activity is physiologically relevant. The context of araL within the arabinose metabolic operon araABDLMNPQ-abfA, as involved in the transport of l-arabinose oligomers, further intracellu- lar degradation and catabolism of l-arabinose [12,28,29], suggests a possible role as a phosphatase active towards sugar phosphate intermediates in l- arabinose catabolism, such as d-xylulose 5-phosphate. On the basis of this, as well as the observation that many HAD members display phosphatase activities against various intermediates of the central metabolic pathways, glycolysis and the pentose phosphate path- way [3], we tested AraL activity towards glucose 6- phosphate, fructose 6-phosphate, fructose 1,6-bisphos- phate, 3-phosphoglycerate, ribose 5-phosphate, d-xylu- lose 5-phosphate and galactose 1-phosphate. Although, B. subtilis does not utilize d-arabinose, the activity towards d-arabinose 5-phosphate was also assayed. In addition, the nucleotides AMP, ADP, ATP, pyri- doxal 5-phosphate and thiamine monophosphate were also screened (Table 2). Although the optimal tempera- ture for enzyme activity is 65 C, the kinetics parame- ters were measured at 37 C, which is the optimal growth temperature for B. subtilis. It is noteworthy that, under these conditions, the KM determined for pNPP is 50 mm (Table 2) compared to 3 mm obtained at 65 C (data not shown). The AraL enzyme showed reactivity with d-xylu- lose 5-phosphate, d-arabinose 5-phosphate, galactose 1-phosphate, glucose 6-phosphate, fructose 6-phos- phate and fructose 1,6-bisphosphate (Table 2). The KM values are high ( 30 mm) and above the range of the known bacterial physiological concentrations. In E. coli, the intracellular concentration of ribose 5-phos- phate, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate is in the range 0.18–6 mm [3] and, in B. subtilis, the measured concentration of fructose 1,6-bisphosphate when cells were grown in the presence of different carbon sources, including arabi- nose, varies in the range 1.8–14.1 mm [30]. However, we cannot rule them out as feasible physiological substrates because, under certain conditions, the intracellular concentrations of glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate may reach 20–50 mm, as reported for Lactococcus lactis [31]. Nev- ertheless, the mean value of the substrate speciﬁcity constant kcat ⁄ KM is low (1 · 102 m)1Æs)1); thus, the abil- ity of AraL to distinguish between these sugar phos- phate substrates will be limited. The results obtained for AraL are comparable to those obtained for other members of HAD from subfamilies IIA and IIB, which have in common a low substrate speciﬁcity and catalytic efﬁciencies (kcat ⁄ KM < 1 · 105 m)1Æs)1) and 2516 FEBS Journal 278 (2011) 2511–2524 ª 2011 The Authors Journal compilation ª 2011 FEBS