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 ϳ4.4 mol of zinc per mol of enzyme. The homotetrameric enzyme had K m and V max values of 1.0 mM and 2.5 mol/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.In a biological system, pyrimidines are metabolized through either a reductive or an oxidative pathway (1, 2). It is well recognized that mammals, plants, and microorganisms utilize the reductive pathway for pyrimidine degradation (3-5), whereas some microorganisms use the oxidative pathway (6 -8).In reductive pyrimidine metabolism, uracil, or thymine is first reduced to its dihydro-derivative, which in turn is hydrolyzed to an N-carbamoyl-␤-amino acid and finally decarbamoylated to a ␤-amino acid. This metabolic route, especially the hydrolysis of dihydro-derivatives catalyzed by dihydropyrimidinase, has attracted much attention, because it is a potential target for drug therapy in the treatment of cancer (9, 10), and it also has been used for the industrial production of optically active amino acids (5,11,12). In contrast, oxidative pyrimidine metabolism has been scarcely investigated, and the references available so far are limited to the early studies performed by three groups of scientists (6 -8). These reports showed that pyrimidine bases are first oxidized to barbituric acid derivatives, and then the barbituric acid derivatives are further hydrolyzed by barbiturase (EC 3.5.2.1) to urea and malonate derivatives. However, these studies were carried out with crude enzyme preparations, and the results presented were inadequate for confirming the enzymatic conversion of barbituric acid to urea and malonate.We have elucidated the enzymes involved in the oxidative pathway, and clarified their physiological functions, in a ...

The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems

“…In both the reductive and oxidative pathways for pyrimidine catabolism described previously (22,48,52) the N-3-C-4 bond is cleaved hydrolytically after the C-5-C-6 double bond has been altered to decrease the aromaticity of the ring (Fig. 1).…”

“…Comparison of Rut pathway products (E. coli K-12) to those of other pyrimidine catabolic pathways. (A) The Rut pathway, which has been studied only in vivo in E. coli K-12 (31); (B) known reductive (52) and oxidative (22,28,48) pathways for catabolism of pyrimidine rings (upper and lower pathways, respectively). Although the enzyme that initiates the oxidative pathway was originally called uracil oxidase, it is a classical monooxygenase (28).…”

“…In the known reductive and oxidative pathways for degradation of the pyrimidine ring (22,48,52), the C-5-C-6 double bond is first altered to decrease the aromatic character of the ring, and it is then hydrolyzed between N-3 and C-4 ( Fig. 1).…”

Defining Sequence Space and Reaction Products within the Cyanuric Acid Hydrolase (AtzD)/Barbiturase Protein Family

“…AtzD and TrzD were identified as being homologous, but they were not connected to any other proteins or protein families in those studies (12,20,26). Barbiturase was first purified to homogeneity from Rhodococcus erythropolis JCM3132 and shown to catalyze the hydrolytic opening of barbituric acid, an intermediate in the oxidative pyrimidine catabolic pathway (34,35). Those studies reported that barbiturase was homologous to AtzD and TrzD (34,35).…”

“…Barbiturase was first purified to homogeneity from Rhodococcus erythropolis JCM3132 and shown to catalyze the hydrolytic opening of barbituric acid, an intermediate in the oxidative pyrimidine catabolic pathway (34,35). Those studies reported that barbiturase was homologous to AtzD and TrzD (34,35). One of the reports found zinc to be present in the enzyme preparations and thus proposed that barbiturase was homologous to members of the amidohydrolase superfamily (35).…”