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Partial Restoration of Activity to Lactobacillus casei Thymidylate Synthase following Inactivation by Domain Deletion

Schellenberger, U and Balaram, P and Francis, VSNK and Shoichet, BK and Santi, Daniel V (1994) Partial Restoration of Activity to Lactobacillus casei Thymidylate Synthase following Inactivation by Domain Deletion. In: Biochemistry, 33 (18). pp. 5623-5629.

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Thymidylate synthase (TS) from Lactobacillus casei has a 50 amino acid insert (residues 90-1 39) in the small domain that is found in only one other TS. A deletion mutant was constructed which lacked the entire insert, thereby reducing the small domain to the size found in Escherichia 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 activity might have resulted from the exposure of residues of helices C and D, which were previously buried by the insert. To restore the local structure of helices C and D in the deletion mutants, we replaced several 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 activity. The mutant which had all those mutations except F87D did not catalyze any dTMP formation. The crucial role of F87D was proven in a deletion 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 parameters for dTMP formation of the active mutants show that the deletion has its major effect on kcat and binding of cofactor CHzHdfolate, with less effect on binding of the substrate dUMP. Removal of residues 90-139 is believed to disorder helices C and D, which in turn decreases cofactor binding and catalysis.

Item Type: Journal Article
Additional Information: Copyright for this article belongs to American Chemical Society.
Department/Centre: Division of Biological Sciences > Molecular Biophysics Unit
Depositing User: M.K Anitha
Date Deposited: 11 Nov 2004
Last Modified: 19 Sep 2010 04:17
URI: http://eprints.iisc.ac.in/id/eprint/2315

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