The Protein Journal

, Volume 29, Issue 6, pp 445–452

Identification and Characterization of a Putative Dihydroorotase, KPN01074, from Klebsiella pneumoniae

  • Chuan-Cheng Wang
  • Huai-Wen Tsau
  • Wei-Ti Chen
  • Cheng-Yang Huang
Article

DOI: 10.1007/s10930-010-9272-2

Cite this article as:
Wang, CC., Tsau, HW., Chen, WT. et al. Protein J (2010) 29: 445. doi:10.1007/s10930-010-9272-2

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 Km = 0.04 mM and Vmax = 8.87 μmol/(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.

Keywords

Dihydroorotase Dihydropyrimidinase Metalloenzyme CAD Hydantoinase 

Abbreviations

DHO

Dihydroorotase

CPS

Carbamoyl phosphate synthetase

ATC

Aspartate transcarbamoylase

Kp

Klebsiella pneumoniae

Ec

Escherichia coli

Aae

Aquifex aeolicus

Dra

Deinococcus radiophilus

CAD

Carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase protein

Km

Michaelis-Menten constant

SDS–PAGE

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis

Vmax

Maximum rate of conversion of an enzyme catalyzed reaction

EDTA

Ethylenediamine tetraacetic acid

Pa

Pseudomonas aeruginosa

St

Salmonella typhimurium

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Chuan-Cheng Wang
    • 1
  • Huai-Wen Tsau
    • 2
  • Wei-Ti Chen
    • 2
  • Cheng-Yang Huang
    • 2
    • 3
  1. 1.Division of Hematology-Oncology, Department of Internal MedicineChanghua Christian HospitalChanghua CityTaiwan
  2. 2.Department of Biomedical SciencesChung Shan Medical UniversityTaichung CityTaiwan
  3. 3.Department of Medical ResearchChung Shan Medical University HospitalTaichung CityTaiwan