Barbiturase, a novel zinc-containing amidohydrolase involved in oxidative pyrimidine metabolism
| Autor: | Chee-Leong Soong, Jun Ogawa, Eiji Sakuradani, Sakayu Shimizu |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Pyrimidine
Stereochemistry Amino Acid Motifs Molecular Sequence Data Biochemistry Binding Competitive Amidohydrolases chemistry.chemical_compound Aminohydrolases Barbiturase Escherichia coli Rhodococcus Amino Acid Sequence Cloning Molecular Molecular Biology Phylogeny chemistry.chemical_classification Barbituric acid Binding Sites Amidohydrolase Base Sequence Sequence Homology Amino Acid Triazines Cyanuric acid amidohydrolase Temperature Cell Biology Hydrogen-Ion Concentration Malonates Recombinant Proteins Oxygen Kinetics Zinc Enzyme Pyrimidines chemistry Models Chemical Spectrophotometry Pyrimidine metabolism Electrophoresis Polyacrylamide Gel Cyanuric acid Peptides |
| Zdroj: | The Journal of biological chemistry. 277(9) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|