title: Identification and characterization of a putative dihydroorotase, KPN01074, from Klebsiella pneumoniae.
abstract: Dihydroorotase (DHO; EC 3.5.2.3) is an essential metalloenzyme in the biosynthesis of pyrimidine nucleotides. Here, we identified and characterized DHO from the pathogenic bacterium Klebsiella pneumoniae (Kp). The activity of KpDHO toward L: -dihydroorotate was observed with K (m) = 0.04 mM and V (max) = 8.87 mumol/(mg min). Supplementing the standard growth medium with Co2+, Mn2+, Mg2+, or Ni2+ increased enzyme activity. The catalytic activity of KpDHO was inhibited with Co2+, Zn2+, Mn2+, Cd2+, Ni2+, and phosphate ions. Substituting the putative metal binding residues His17, His19, Lys103, His140, His178, and Asp251 with Ala completely abolished KpDHO activity. However, the activity of the mutant D251E was fourfold higher than that of the wild-type protein. On the basis of these biochemical and mutational analyses, KpDHO (KPN01074) was identified as type II DHO.
authors.affiliation: Division of Hematology-Oncology, Department of Internal Medicine, Changhua Christian Hospital, No. 135, Nanxiao St., Changhua City, Taiwan.
authors.forename: Chuan-Cheng
authors.initials: CC
authors.lastname: Wang
authors.forename: Huai-Wen
authors.initials: HW
authors.lastname: Tsau
authors.forename: Wei-Ti
authors.initials: WT
authors.lastname: Chen
authors.forename: Cheng-Yang
authors.initials: CY
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004080:Dihydroorotase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D019216:Metals, Heavy / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D019216:Metals, Heavy; D004080:Dihydroorotase