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

Rahul Shah, Catherine S. Coleman, Kiran Mir, Jeffrey Baldwin, James L. Van Etten, Nick V. Grishinll, Anthony E. Pegg, Bruce A. Stanley, Margaret A. Phillips

Research output: Contribution to journalArticle

27 Citations (Scopus)

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 δ-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 kcat/Km for L-arginine is 550-fold higher than for either L-ornithine or L-lysine, which were decarboxylated with similar efficiency. In addition, α- difluoromethylarginine was a more potent inhibitor of the enzyme than α-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 re-classified 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 β/α-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.

Original languageEnglish (US)
Pages (from-to)35760-35767
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number34
DOIs
StatePublished - Aug 20 2004

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Paramecium
Chlorella
Ornithine
Ornithine Decarboxylase
Viruses
Carboxy-Lyases
Arginine
Substrates
Substrate Specificity
Catalytic Domain
Substitution reactions
Eflornithine
Basic Amino Acids
Trypanosoma brucei brucei
Chlorophyta
DNA Viruses
Enzyme Inhibitors
Amino Acid Substitution
Algae
Lysine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Paramecium bursaria chlorella virus-1 encodes an unusual arginine decarboxylase that is a close homolog of eukaryotic ornithine decarboxylases. / Shah, Rahul; Coleman, Catherine S.; Mir, Kiran; Baldwin, Jeffrey; Van Etten, James L.; Grishinll, Nick V.; Pegg, Anthony E.; Stanley, Bruce A.; Phillips, Margaret A.

In: Journal of Biological Chemistry, Vol. 279, No. 34, 20.08.2004, p. 35760-35767.

Research output: Contribution to journalArticle

Shah, Rahul ; Coleman, Catherine S. ; Mir, Kiran ; Baldwin, Jeffrey ; Van Etten, James L. ; Grishinll, Nick V. ; Pegg, Anthony E. ; Stanley, Bruce A. ; Phillips, Margaret A. / Paramecium bursaria chlorella virus-1 encodes an unusual arginine decarboxylase that is a close homolog of eukaryotic ornithine decarboxylases. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 34. pp. 35760-35767.
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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 δ-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 kcat/Km for L-arginine is 550-fold higher than for either L-ornithine or L-lysine, which were decarboxylated with similar efficiency. In addition, α- difluoromethylarginine was a more potent inhibitor of the enzyme than α-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 re-classified 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 β/α-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.",
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AU - Baldwin, Jeffrey

AU - Van Etten, James L.

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AU - Stanley, Bruce A.

AU - Phillips, Margaret A.

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