Article Figures & Data
Figures
- FIG. 1.
Phylogenic analysis of various IMPases. For each protein, the corresponding organisms and the accession number from the databanks are shown. The full names of the species under study, except those already mentioned in the text or the figure, were Agrobacterium tumefaciens C58, Aquifex aeolicus VF5, Brucella melitensis 16M, Caenorhabditis elegans, Cytophaga hutchinsonii, Desulfitobacterium hafniense, Gloeobacter violaceus PCC7421, Magnetospirillum magnetotacticum, Mesembryanthemum crystallinum, Mesorhizobium loti MAFF303099, Methylobacillus flagellatus KT, Mycobacterium tuberculosis, Neurospora crassa, Prochlorococcus marinus MIT9313, Prochlorococcus marinus CCMP1375, Prochlorococcus marinus CCMP1986, Pseudomonas syringae DC3000, Rhodobacter sphaeroides, Rhodospirillum rubrum, Streptomyces coelicolor A3(2), Synechococcus elongatus PCC6301, Thermosynechococcus elongatus BP-1, Xanthomonas axonopodis 306, and Xenopus laevis. Based on this analysis, five different clusters can be clearly defined (clades I through V). Proteins from cyanobacteria are marked with a triangle. Proteins of particular relevance to this work are shown in boldface. Bootstrap values are labeled on the major branches.
- FIG. 2.
Analysis of the purified HalA on an SDS-12% polyacrylamide electrophoresis gel stained with Coomassie brilliant blue. Lane 1, protein extract from E. coli transformed with plasmid pET28a(+) (control); lane 2, protein extract from E. coli transformed with plasmid pET28a-ArHAL (containing the coding region of the halA gene); lane 3, HalA after purification in a HisTrap affinity column; lane 4, molecular mass standards in kilodaltons.
- FIG. 3.
Effects of Mg2+ (A) and pH (B) on the activity of HalA. Error bars indicate standard deviations.
- FIG. 4.
Effects of various ions on the enzyme activity of HalA with 0.5 mM PAP as substrate. The activity was measured in a buffer containing 50 mM Tris, pH 8.0, and 2 mM MgCl2 at 30°C for 40 min. The activity obtained at optimal conditions was considered 100%, and that obtained in the presence of ions was normalized to the optimal activity. Error bars indicate standard deviations.
- FIG. 5.
Effects of the expression of halA in E. coli on the tolerance toward LiCl. E. coli strain BL21 (DE3) was transformed with either pET28a-ArHAL (A and C) or pET28a(+) (B and D). The two transformed strains were grown in M9 medium supplemented with MgSO4 as the sulfur source and 0.04 mM IPTG as the inducer. For A and B, no LiCl was added; for C and D, 0.4 M LiCl was added. The growth rate was followed by measuring the optical density (OD) by spectroscopy at 600 nm. Error bars show the results of three independent experiments.
- FIG. 6.
Modeling of the substrate-binding site of HalA. The model was based on the high-resolution X-ray structure of Hal2p from yeast (2, 19). A major difference between Hal2p and HalA was found at the substrate-binding pocket with the presence of the ring structure of Pro254 in HalA.
Tables
- TABLE 2.
Comparison of different parameters of various enzymes
Enzyme Substrates Sensitivity (IC50) (mM) to: Km for PAP (μM) Family Source or reference Na+ Li+ HalA PAP, PAPS 600 3.6 1,035 PAPase This study Hal2p PAP, PAPS 20 0.1 <20 PAPase 16 AtAHL PAP, PAPS 50 10 160 PAPase 9 Tol1 PAP, PAPS, IBP SMa SMMb >20 PIPase 15 Sal1 PAP, PAPS, IBP 200 0.2 2-10 PIPase 21 Sal2 PAP, PAPS, IBP 200 10 >20 PIPase 9
