The activity of Escherichia coli dihydroorotate dehydrogenase is dependent on a conserved loop identified by sequence homology, mutagenesis, and limited proteolysis

Biochemistry. 1999 Mar 9;38(10):2899-908. doi: 10.1021/bi982352c.


Dihydroorotate dehydrogenase catalyzes the oxidation of dihydroorotate to orotate. The enzyme from Escherichia coli was overproduced and characterized in comparison with the dimeric Lactococcus lactis A enzyme, whose structure is known. The two enzymes represent two distinct evolutionary families of dihydroorotate dehydrogenases, but sedimentation in sucrose gradients suggests a dimeric structure also of the E. coli enzyme. Product inhibition showed that the E. coli enzyme, in contrast to the L. lactis enzyme, has separate binding sites for dihydroorotate and the electron acceptor. Trypsin readily cleaved the E. coli enzyme into two fragments of 182 and 154 residues, respectively. Cleavage reduced the activity more than 100-fold but left other molecular properties, including the heat stability, intact. The trypsin cleavage site, at R182, is positioned in a conserved region that, in the L. lactis enzyme, forms a loop where a cysteine residue is very critical for activity. In the corresponding position, the enzyme from E. coli has a serine residue. Mutagenesis of this residue (S175) to alanine or cysteine reduced the activities 10000- and 500-fold, respectively. The S175C mutant was also defective with respect to substrate and product binding. Structural and mechanistic differences between the two different families of dihydroorotate dehydrogenase are discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Chromatography, Gel
  • Conserved Sequence*
  • Dihydroorotate Dehydrogenase
  • Enzyme Activation / genetics
  • Enzyme Stability / genetics
  • Escherichia coli / enzymology*
  • Hydrolysis
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Kinetics
  • Mutagenesis, Site-Directed*
  • Oxidoreductases / genetics
  • Oxidoreductases / isolation & purification
  • Oxidoreductases / metabolism*
  • Oxidoreductases Acting on CH-CH Group Donors*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid*
  • Serine / genetics
  • Spectrometry, Fluorescence
  • Trypsin / metabolism*


  • Dihydroorotate Dehydrogenase
  • Isoenzymes
  • Recombinant Proteins
  • Serine
  • Oxidoreductases
  • Oxidoreductases Acting on CH-CH Group Donors
  • Trypsin