An essential role for water in an enzyme reaction mechanism: the crystal structure of the thymidylate synthase mutant E58Q

Biochemistry
C R SageR M Stroud

Abstract

A water-mediated hydrogen bond network coordinated by glutamate 60(58) appears to play an important role in the thymidylate synthase (TS) reaction mechanism. We have addressed the role of glutamate 60(58) in the TS reaction by cocrystalizing the Escherichia coli TS mutant E60(58)Q with dUMP and the cofactor analog CB3717 and have determined the X-ray crystal structure to 2.5 A resolution with a final R factor of 15.2% (Rfree = 24.0%). Using difference Fourier analysis, we analyzed directly the changes that occur between wild-type and mutant structures. The structure of the mutant enzyme suggests that E60(58) is not required to properly position the ligands in the active site and that the coordinated hydrogen bond network has been disrupted in the mutant, providing an atomic resolution explanation for the impairment of the TS reaction by the E60(58)Q mutant and confirming the proposal that E60(58) coordinates this conserved hydrogen bond network. The structure also provides insight into the role of specific waters in the active site which have been suggested to be important in the TS reaction. Finally, the structure shows a unique conformation for the cofactor analog, CB3717, which has implications for structure-based drug desig...Continue Reading

References

Jan 1, 1995·Annual Review of Biochemistry·C W Carreras, D V Santi
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May 1, 1993·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·R M Stroud, J S Finer-Moore

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Citations

Jan 7, 1998·Protein Science : a Publication of the Protein Society·P StropW R Montfort
Apr 16, 1998·Pharmacology & Therapeutics·W R Montfort, A Weichsel
Sep 6, 2012·Acta Crystallographica. Section D, Biological Crystallography·Cecilia PozziStefano Mangani
Apr 24, 2001·Protein Science : a Publication of the Protein Society·R AlmogP Van Roey
Mar 26, 1999·Protein Science : a Publication of the Protein Society·K M FoxP Van Roey
Aug 19, 2015·The Journal of Physical Chemistry Letters·Jooyong LeeMinhaeng Cho
Nov 26, 2015·Archives of Biochemistry and Biophysics·William B O'DellFlora Meilleur
May 16, 2006·Proteins·Christopher A BottomsJohn J Tanner
Apr 29, 2015·Physical Chemistry Chemical Physics : PCCP·Ananda K GhoshAmnon Kohen
Aug 4, 1998·Structure·T J StoutR M Stroud
Jun 4, 2021·ACS Central Science·Taku Kitanosono, Shu Kobayashi

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