-
The Biochemical Journal Aug 1985A new purification procedure involving five column-chromatography steps is described for dihydro-orotase (L-5,6-dihydro-orotate amidohydrolase, EC 3.5.2.3) from...
A new purification procedure involving five column-chromatography steps is described for dihydro-orotase (L-5,6-dihydro-orotate amidohydrolase, EC 3.5.2.3) from Clostridium oroticum (A.T.C.C. 25750). The native purified enzyme is a dimer of Mr 102 000 and contains 4.0 +/- 0.3 g-atoms of zinc/mol of dimer. These observations agree with those reported previously [Taylor, Taylor, Balch & Gilchrist (1976) J. Bacteriol. 127, 863-873]. It is conclusively demonstrated that dihydro-orotase is a zinc metalloenzyme. Zinc is reversibly removed by treatment with chelators in phosphate buffer at pH 6.5, as demonstrated by atomic absorption spectrophotometry and decrease of enzyme activity. The specific activity is linearly dependent on zinc content. Addition of ZnSO4 to the chelator-treated enzyme results in regain of the normal complement of zinc and enzyme activity. Kinetic properties of the reconstituted enzyme are indistinguishable from those of the native enzyme. The amino acid composition of the homogeneous enzyme suggests that the zinc atoms occupy different environments.
Topics: Amidohydrolases; Amino Acids; Chromatography, Liquid; Clostridium; Dihydroorotase; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Kinetics; Zinc
PubMed: 2864918
DOI: 10.1042/bj2300101 -
Journal of Bacteriology Aug 1976Dihydroorotase +4,5-L-dihydro-orotate amidohydrolase [EC 3.5.2.3]), which catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate, has been...
Dihydroorotase +4,5-L-dihydro-orotate amidohydrolase [EC 3.5.2.3]), which catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate, has been purified from orotate-grown Clostridium oroticum. The enzyme is homogeneous when subjected to polyacrylamide gel electrophoresis and is stable at pH 7.6 in 0.3 M NaCl containing 10 muM ZnSO4. The enzyme has a molecular weight of approximately 110,000. Sodium dodecyl sulfate gel electrophoresis, using three different buffer systems, indicated the enzyme is composed of two subunits, each having a molecular weight of 55,000. Dihydroorotase is shown by atomic absorption spectroscopy to be a zinc-containing metalloenzyme with 4 g-atoms of zinc per 110,000 g of protein. The pH optima for the conversion of N-carbamyl-L-aspartate to L-dihydroorotate and for L-dihydroorotate to N-carbamyl-L-aspartate are pH 6.0 and 8.2, respectively. The Km values for N-carbamyl-L-aspartate and for L-dihydroorotate are 0.13 and 0.07 mM, respectively. Inhibitor studies indicate that zinc may be involved in the catalytic activity of the enzyme.
Topics: Amidohydrolases; Aspartic Acid; Cell-Free System; Clostridium; Edetic Acid; Hydrogen-Ion Concentration; Metalloproteins; Molecular Weight; Orotic Acid; Phenanthrolines; Phosphates; Zinc
PubMed: 8424
DOI: 10.1128/jb.127.2.863-873.1976 -
Microbiology and Immunology 2006Screening of faecal bacteria for glycyrrhetic acid (GA) production by hydrolysing of glycyrrhizin (GL) resulted in the isolation of two strains, designated ZM35T and...
Screening of faecal bacteria for glycyrrhetic acid (GA) production by hydrolysing of glycyrrhizin (GL) resulted in the isolation of two strains, designated ZM35T and ZM38. Strains ZM35T and ZM38 were Gram-positive, obligate anaerobic, non-spore-forming and rod-shaped bacteria. Analysis of the 16S rRNA gene sequences indicated that strains ZM35T and ZM38 belonged to cluster XIVa of the genus Clostridium. The 16S rRNA gene sequences of strains ZM35T and ZM38 were identical. Strain ZM35T exhibited approximately 94% to 95% identity with the validly described species, Clostridium oroticum(94.5%), Eubacterium contortum(93.8%), Ruminococcus gnavus(94.5%) and R. torques(95.1%). In an experiment of DNA-DNA hybridization, it was confirmed that strains ZM35T and ZM38 were the same species. The guanine-plus-cytosine (G+C) content of strain ZM35T is 45.7 mol%. Based on the phylogenetic and phenotypic findings, we propose that strains ZM35T and ZM38 be assigned to a novel species named Clostridium glycyrrhizinilyticum. The type strain is ZM35T (=JCM 13368T=DSM 17593T).
Topics: Clostridium; Feces; Glycyrrhetinic Acid; Glycyrrhizic Acid; Humans; Hydrolysis; Molecular Sequence Data; Phylogeny; RNA, Ribosomal, 16S
PubMed: 16858139
DOI: 10.1111/j.1348-0421.2006.tb03818.x -
New Microbes and New Infections Mar 2021An anaerobic facultative Gram-stain positive bacterium was isolated from human gut microbiota. Strain Marseille-P5551 was considered to be a new genus within the phylum...
An anaerobic facultative Gram-stain positive bacterium was isolated from human gut microbiota. Strain Marseille-P5551 was considered to be a new genus within the phylum , as it exhibits a 91.87% similarity level with (NR_117129.1), the phylogenetically closest related species. The draft genome size of strain Marseille-P5551 is 4 142 938 bp with 44.4% of G + C content. We hereby suggest the creation of gen. nov., sp. nov., as a new bacterial genus.
PubMed: 33732473
DOI: 10.1016/j.nmni.2021.100850