L. M. Godinho and I. de Sa´ -Nogueira AraL sugar phosphatase from B. subtilis Fig. 4. Effect of pH, temperature and co-factor concentration on AraL activity. Enzyme activity was determined using pNPP as substrate, at 65 C, pH 7, and 15 mM MgCl2, unless stated otherwise. The results represent the mean of three independent experiments. Table 2. Kinetic constants for AraL against various substrates. Assays were performed at pH 7 and 37 C, as described in the Experimental procedures. The results are the mean ± SD of tripli- cates. Substrates tested for which no activity was detected were: ATP, ADP, AMP, ribose 5-phosphate, glycerol 3-phosphate, pyri- doxal 5-phosphate and thiamine monophosphate. Substrate KM (mM) kcat (s)1) 0.943 · 102 D-xylulose 29.14 ± 4.87 2.75 ± 0.26 5-phosphate 0.998 · 102 Glucose 24.96 ± 4.08 2.49 ± 0.26 6-phosphate D-Arabinose 27.36 ± 1.8 2.92 ± 0.10 1.06 · 102 5-phosphate 0.807 · 102 Fructose 34.89 ± 4.51 2.817 ± 0.22 6-phosphate 0.365 · 102 Fructose 40.78 ± 11.40 1.49 ± 0.26 1,6-bisphosphate Galactose 1-phosphate 40.74 ± 6.03 4.28 ± 0.40 1.02 · 102 pNPP 50.00 ± 23.32 0.012 ± 0.0006 0.24 kcat ⁄ KM (s)1ÆM)1) lack deﬁned boundaries of physiological substrates [10,13]. These features are indicative of enzymes func- tioning in secondary metabolic pathways. Production of AraL in E. coli is subjected to regulation In silico DNA sequence analysis of pLG12 and pLG5 detected the possible formation, in both plasmids, of a mRNA secondary structure, which sequesters the ribosome-binding site. Both, hairpin structures, display a low free energy of )17.5 kcalÆmol)1 (Fig. 5A) and )22.7 kcalÆmol)1 (data not shown), respectively, and could impair translation that pre- vents the production of AraL observed in these con- structs (see above). In plasmid pLG11 carrying the truncated version of AraL, overproduction was suc- cessful (Fig. 3). Deletion of the 5¢-end of the araL gene caused an increase of the free energy of the putative mRNA secondary structure ()11.8 kcalÆmol)1; data not shown). To test the potential involvement of the mRNA secondary structure in the lack of production of the recombinant AraL ver- sions constructed in plasmids pLG12 and pLG5, site-directed mutagenesis was performed using pLG12 as template. A single-base substitution G ﬁ A intro- duced at the 5¢-end of the gene (Fig. 1) was designed to increase the free energy of the mRNA secondary structure in the resulting plasmid pLG13. This point mutation increased the free energy from )17.5 kcalÆmol)1 to )13.1 kcalÆmol)1 (Fig. 5A). In addition, this modiﬁcation caused the substitution of a glycine to an aspartate at position 12 in AraL (G12 ﬁ D; Fig. 1); however, based on the structure of NagD from E. coli [13], this amino acid substitu- tion close to the N-terminus is not expected to cause major interference in the overall protein folding. Cell extracts of induced E. coli Bl21 pLys(S) DE3 cells carrying pLG13 were tested for the presence of AraL. A strong band with an estimated size of 29 kDa was detected (Fig. 5B), strongly suggesting that recombinant AraL is produced in E. coli when the mRNA secondary structure is destabilized. This observation indicates that the production of AraL is modulated by a secondary mRNA structure placed at the 5¢-end of the araL gene. FEBS Journal 278 (2011) 2511–2524 ª 2011 The Authors Journal compilation ª 2011 FEBS 2517