Paramecium bursaria chlorella virus-1 encodes an unusual arginine decarboxylase that is a close homolog of eukaryotic ornithine decarboxylases

J Biol Chem. 2004 Aug 20;279(34):35760-7. doi: 10.1074/jbc.M405366200. Epub 2004 Jun 9.

Abstract

Paramecium bursaria chlorella virus (PBCV-1) is a large double-stranded DNA virus that infects chlorella-like green algae. The virus encodes a homolog of eukaryotic ornithine decarboxylase (ODC) that was previously demonstrated to be capable of decarboxylating l-ornithine. However, the active site of this enzyme contains a key amino acid substitution (Glu for Asp) of a residue that interacts with the delta-amino group of ornithine analogs in the x-ray structures of ODC. To determine whether this active-site change affects substrate specificity, kinetic analysis of the PBCV-1 decarboxylase (PBCV-1 DC) on three basic amino acids was undertaken. The k(cat)/K(m) for l-arginine is 550-fold higher than for either l-ornithine or l-lysine, which were decarboxylated with similar efficiency. In addition, alpha-difluoromethylarginine was a more potent inhibitor of the enzyme than alpha-difluoromethylornithine. Mass spectrometric analysis demonstrated that inactivation was consistent with the formation of a covalent adduct at Cys(347). These data demonstrate that PBCV-1 DC should be reclassified as an arginine decarboxylase. The eukaryotic ODCs, as well as PBCV-1 DC, are only distantly related to the bacterial and plant arginine decarboxylases from their common beta/alpha-fold class; thus, the finding that PBCV-1 DC prefers l-arginine to l-ornithine was unexpected based on evolutionary analysis. Mutational analysis was carried out to determine whether the Asp-to-Glu substitution at position 296 (position 332 in Trypanosoma brucei ODC) conferred the change in substrate specificity. This residue was found to be an important determinant of substrate binding for both l-arginine and l-ornithine, but it is not sufficient to encode the change in substrate preference.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Carboxy-Lyases / genetics*
  • Carboxy-Lyases / metabolism
  • Chlorella / virology*
  • DNA Mutational Analysis
  • DNA, Viral
  • Genes, Viral
  • Molecular Sequence Data
  • Ornithine Decarboxylase / genetics
  • Phycodnaviridae / enzymology*
  • Phycodnaviridae / genetics
  • Sequence Alignment
  • Sequence Homology
  • Substrate Specificity
  • Viral Proteins / genetics
  • Viral Proteins / metabolism

Substances

  • DNA, Viral
  • Viral Proteins
  • Carboxy-Lyases
  • Ornithine Decarboxylase
  • arginine decarboxylase