title: Structure-based alteration of substrate specificity and catalytic activity of sulfite oxidase from sulfite oxidation to nitrate reduction.
abstract: Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conserved in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 Å resolution, respectively.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, United States.
authors.forename: James A
authors.initials: JA
authors.lastname: Qiu
authors.forename: Heather L
authors.initials: HL
authors.lastname: Wilson
authors.forename: K V
authors.initials: KV
authors.lastname: Rajagopalan
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D017360:Arabidopsis; D020134:Catalytic Domain; D002645:Chickens; D018360:Crystallography, X-Ray; D014644:Genetic Variation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D050876:Sulfite Oxidase / Q000737:chemistry* / Q000235:genetics; D013447:Sulfites / Q000737:chemistry* / Q000378:metabolism
chemical_list: D013447:Sulfites; D050901:Nitrate Reductase; D050876:Sulfite Oxidase