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.
Highlights
Paramecium bursaria chlorella virus-1 (PBCV-1)1 is a large, icosahedral, plaque-forming, double-stranded DNA virus that replicates in certain unicellular, eukaryotic, chlorella-like green algae [1, 2]
All enzymes in the family share a number of essential active-site residues (24, 29 –32), eukaryotic ornithine decarboxylases (ODCs) are very distantly related to arginine decarboxylase (ADC) and diaminopimelate decarboxylase and share only ϳ15% overall sequence identity based on pairwise comparisons
The pyridoxal 5Ј-phosphate (PLP)-dependent decarboxylases that belong to the /␣barrel fold are from both bacterial and eukaryotic origins, and they include enzymes capable of decarboxylating a range of basic amino acid substrates [20]
Summary
Materials—Amino acids, polyamines, and a carbon dioxide kit were purchased from Sigma. Spectroscopy-based Enzymatic Assays—The steady-state kinetics of the decarboxylation of L-ornithine (1–25 mM), L-arginine (0.5-l6 mM), or L-lysine (1–50 mM) by PBCV-1 DC, TbODC, PBCV-1 DC E296D, or TbODC D332E were measured spectrophotometrically at 37 °C using a Sigma diagnostics carbon dioxide detection kit as described previously [36]. HPLC Analysis of Reaction Products—The products of an enzymatic reaction with L-arginine and wild-type PBCV-1 DC and TbODC D332E were analyzed by HPLC using an AccQ-Tag kit (Waters) in 5% sodium tetraborate and labeling reagent (6-aminoquinolyl-n-hydroxysuccinimidyl in acetonitrile) as described previously [24]. Samples were digested with Glu-C in 25 mM NH4HCO3 (pH 7.8) containing 10% (v/v) acetonitrile for 2–16 h at 25 °C, stopped by the addition of glacial acetic acid, and stored frozen until analysis by MALDI-TOF using an Applied Biosystems 4700 Proteomics Analyzer. Each spot was overlaid with 0.6 l of freshly prepared matrix (5 mg/ml recrystallized ␣-cyanohydroxycinnamic acid, 2 mg/ml NH4H2PO4, 50% acetonitrile, and 0.1% trifluoroacetic acid)
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