Characterization of heterosubunit complexes formed by the R1 and R2 subunits of herpes simplex virus 1 and equine herpes virus 4 ribonucleotide reductase.
We report on the separate PCR cloning and subsequent expression and purification of the large (R1) and small (R2) subunits from equine herpes virus type 4 (EHV-4) ribonucleotide reductase. The EHV-4 R1 and R2 subunits reconstituted an active enzyme and their abilities to complement the R1 and R2 subunits from the closely related herpes simplex virus 1 (HSV-1) ribonucleotide reductase, with the use of subunit interaction and enzyme activity assays, were analysed. Both EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 were able to assemble heterosubunit complexes but, surprisingly, neither of these complexes was fully active in enzyme activity assays; the EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 enzymes had 50% and 5% of their respective wild-type activities. Site-directed mutagenesis was used to alter two non-conserved residues located within the highly conserved and functionally important C-termini of the EHV-4 and HSV-1 R1 proteins. Mutation of Pro-737 to Lys and Lys-1084 to Pro in EHV-4 and HSV-1 R1 respectively had no effects on subunit assembly. Mutation of Pro-737 to Lys in EHV-4 R1 decreased enzyme activity by 50%; replacement of Lys-1084 by Pro in HSV-1 R1 had no effect on enzyme activity. Both alterations failed to restore full enzyme activities to the heterosubunit enzymes. Therefore probably neither of these amino acids has a direct role in catalysis. However, mutation of the highly conserved Tyr-1111 to Phe in HSV-1 R1 inactivated enzyme activity without affecting subunit interaction.
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School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 0BA, U.K. j.conner@gcal.ac.uk
Conner
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J
D003001:Cloning, Molecular; D019976:Cloning, Organism; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D018140:Varicellovirus / Q000201:enzymology*
D046911:Macromolecular Substances; D009842:Oligopeptides; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases