Structural and functional analysis of surface domains unique to bacteriophage T4 thymidylate synthase.

Abstract

The bacteriophage T4 genome encodes most of its own enzymes for dNTP synthesis, which form a complex in infected Escherichia coli. The T4 thymidylate synthase (TS) and the T4 deoxycytidylate deaminase (CD) catalyze sequential reactions and are physically linked within this complex [McGaughey, K. M., Wheeler, L. J., Moore, J. T., Maley, G. F. , Maley, F., and Mathews, C. K. (1996) J. Biol. Chem. 271, 23037-23042]. From the crystal structure of T4TS [Finer-Moore, J. S., Maley, G. F., Maley, F., Montfort, W. R., and Stroud, R. M. (1994) Biochemistry 33, 15459-15468], it appears that three regions corresponding to insertions relative to E. coli TS lie on one side of the enzyme surface. We have investigated the residual activity of T4TS in response to complete deletion or substitution mutagenesis of these insertions. Two deletions generated in the small domain (residues 70-103) reduced the TS activity to 0.2% and 0.7% of the wild-type activity, with the latter able to complement growth of a thyA- E. coli strain in the absence of thymidine. By insertion of exogenous sequences variable in length and in sequence into these deletion mutants, enzyme activity increased to 44% that of the wild type. Restoration of the TS activity depended mostly on the hydrophobicity of the inserted residues. The sites of insertions also displayed distinct permissiveness for accommodating the exogenous insertions. Deletions and substitutions near the C-terminus resulted in complete inactivation of the T4TS. Proteolysis experiments revealed that the modified surface loops of the small domain were still accessible and flexible for protein-protein interactions. We have used ELISA to detect a physical association between T4TS and T4CD and compared the binding affinity of WT T4TS for two purified insertion mutants of T4CD. The results obtained showed that the native sequences of the small domain inserts are not required for T4TS/T4CD complex formation.