title: A rice HAL2-like gene encodes a Ca(2+)-sensitive 3'(2'),5'-diphosphonucleoside 3'(2')-phosphohydrolase and complements yeast met22 and Escherichia coli cysQ mutations.
abstract: A plant homolog of yeast HAL2 gene (RHL) was cloned from rice (Orizya sativa L.). The RHL cDNA complemented an Escherichia coli cysteine auxotrophic mutant, cysQ, and the yeast HAL2 mutant, met22. The latter is a methionine auxotroph and cannot use sulfate, sulfite, or sulfide as sulfur sources but exhibits wild-type activities of the enzymes necessary to assimilate sulfate and has normal sulfur uptake system. These results demonstrated that HAL2, cysQ, and RHL genes encode proteins with similar function in sulfur assimilatory pathway. The RHL cDNA expressed a 40-kDa protein that was shown to catalyze the conversion of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to adenosine 5'-phosphosulfate (APS) and 3'(2')-phosphoadenosine 5'-phosphate (PAP) to AMP. The enzyme activity is Mg(2+)-dependent, sensitive to Ca2+, Li+, and Na+ and activated by K+. The inhibition by Ca2+ depends on the Mg2+/Ca2+ ratio and is reversible by high Mg2+ concentration. The substrate specificity and kinetics of RHL enzyme are very similar to the Chlorella 3'(2'),5'-diphosphonucleoside 3'(2')-phosphohydrolase (DPNPase). Our evidence suggests that this enzyme regulates the flux of sulfur in the sulfur-activation pathway by converting PAPS to APS. Several residues that are essential for the activity of this enzyme were identified by site-directed mutagenesis, and the possible role of DPNPase in salt tolerance is discussed.
mesh_terms: D000250:Adenosine Phosphosulfate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009708:Nucleotidases / Q000235:genetics*; D012275:Oryza / Q000235:genetics*; D012965:Sodium Chloride / Q000494:pharmacology; D013455:Sulfur / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D012965:Sodium Chloride; D000250:Adenosine Phosphosulfate; D013455:Sulfur; D009708:Nucleotidases; C053354:bisphosphoadenylate 3'-nucleotidase; D008274:Magnesium; D002118:Calcium