PARTIAL RESTORATION OF ACTIVITY TO LACTOBACILLUS-CASEI THYMIDYLATE SYNTHASE FOLLOWING INACTIVATION BY DOMAIN DELETION

Thymidylate synthase (TS) from Lactobacillus casei has a 50 amino acid insert (residues 90-139) in the small domain that is found in only on e other TS. A deletion mutant was constructed which lacked the entire insert, thereby reducing the small domain to the-size found in Escheri chia coli TS. This mutant did not catalyze the formation of dTMP. From the crystal structure of L. casei TS, we surmised that the loss of ac tivity might have resulted from the exposure of residues of helices C and D, which were previously buried by the insert. To restore the loca l structure of helices C and D in the deletion mutants, we replaced se veral residues in this region by the corresponding residues found in E . coli TS. The mutant whose sequence most closely resembled that of E. coli TS carried six mutations and possessed partially restored TS act ivity. The mutant which had all those mutations except F87D did not ca talyze any dTMP formation. The crucial role of F87D was proven in a de letion mutant which had only this change and showed greatly increased activity. All of the mutants catalyzed the debromination of BrdUMP in the absence of cofactor about as well as wild type TS. The kinetic par ameters for dTMP formation of the active mutants show that the deletio n has its major effect on k(cat) and binding of cofactor CH(2)H(4)fola te, with less effect on binding of the substrate dUMP. Removal of resi dues 90-139 is believed to disorder helices C and D, which in turn dec reases cofactor binding and catalysis.