title: Creation of a putative third metal binding site in type II dihydroorotases significantly enhances enzyme activity.
abstract: Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides. DHOase is divided into two types (I and II). Type II DHOase generally contains a binuclear metal center in its active site. Recently, the crystal structure of DHOase domain in human CAD protein (huDHOase) has revealed three metal ions in the protein's active site. However, whether type II DHOase can have the critical third metal ion, as observed in huDHOase, remains unknown. In the present study, the putative third metal binding site in type II enzymes, such as the prokaryotic Salmonella enterica serovar Typhimurium LT2 DHOase (StDHOase) and the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase), was created and identified. StDHOase T198E and ScDHOase T208E mutants had higher activities compared with their wild-type enzymes. The need for a higher DHOase stability and activity may drive creation of the third metal ion binding site in huDHOase, which can be achieved by mutating a highly conserved position T in type II dihydroorotases to E, similar to that in huDHOase.
authors.forename: Yen-Hua
authors.initials: YH
authors.lastname: Huang
authors.affiliation: School of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan, and Department of Medical Research, Chung Shan Medical University Hospital, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan. cyhuang@csmu.edu.tw.
authors.forename: Cheng-Yang
authors.initials: CY
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004080:Dihydroorotase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019779:Salmonella enterica / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C033626:CAD trifunctional enzyme; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)