title: Barbiturase, a novel zinc-containing amidohydrolase involved in oxidative pyrimidine metabolism.
abstract: Barbiturase, which catalyzes the reversible amidohydrolysis of barbituric acid to ureidomalonic acid in the second step of oxidative pyrimidine degradation, was purified to homogeneity from Rhodococcus erythropolis JCM 3132. The characteristics and gene organization of barbiturase suggested that it is a novel zinc-containing amidohydrolase that should be grouped into a new family of the amidohydrolases superfamily. The amino acid sequence of barbiturase exhibited 48% identity with that of herbicide atrazine-decomposing cyanuric acid amidohydrolase but exhibited no significant homology to other proteins, indicating that cyanuric acid amidohydrolase may have evolved from barbiturase. A putative uracil phosphoribosyltransferase gene was found upstream of the barbiturase gene, suggesting mutual interaction between pyrimidine biosynthesis and oxidative degradation. Metal analysis with an inductively coupled radiofrequency plasma spectrophotometer revealed that barbiturase contains approximately 4.4 mol of zinc per mol of enzyme. The homotetrameric enzyme had K(m) and V(max) values of 1.0 mm and 2.5 micromol/min/mg of protein, respectively, for barbituric acid. The enzyme specifically acted on barbituric acid, and dihydro-l-orotate, alloxan, and cyanuric acid competitively inhibited its activity. The full-length gene encoding the barbiturase (bar) was cloned and overexpressed in Escherichia coli. The kinetic parameters and physicochemical properties of the cloned enzyme were apparently similar to those of the wild-type.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Chee-Leong
authors.initials: CL
authors.lastname: Soong
authors.forename: Jun
authors.initials: J
authors.lastname: Ogawa
authors.forename: Eiji
authors.initials: E
authors.lastname: Sakuradani
authors.forename: Sakayu
authors.initials: S
authors.lastname: Shimizu
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000502:physiology*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008314:Malonates / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D011743:Pyrimidines / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D012240:Rhodococcus / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013696:Temperature; D014227:Triazines / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry*
chemical_list: D008314:Malonates; D010455:Peptides; D011743:Pyrimidines; D011994:Recombinant Proteins; D014227:Triazines; C030290:malonic acid; D000581:Amidohydrolases; C434739:barbiturase; D000619:Aminohydrolases; C067141:cyanuric acid amidohydrolase; C004632:cyanuric acid; D015032:Zinc; C030986:pyrimidine; D010100:Oxygen