Format

Send to

Choose Destination
J Am Chem Soc. 2012 Mar 7;134(9):4442-8. doi: 10.1021/ja2120822. Epub 2012 Feb 24.

Trapping of an intermediate in the reaction catalyzed by flavin-dependent thymidylate synthase.

Author information

1
Department of Chemistry, The University of Iowa, Iowa City, Iowa 52242, USA.

Abstract

Thymidylate is a DNA nucleotide that is essential to all organisms and is synthesized by the enzyme thymidylate synthase (TSase). Several human pathogens rely on an alternative flavin-dependent thymidylate synthase (FDTS), which differs from the human TSase both in structure and molecular mechanism. It has recently been shown that FDTS catalysis does not rely on an enzymatic nucleophile and that the proposed reaction intermediates are not covalently bound to the enzyme during catalysis, an important distinction from the human TSase. Here we report the chemical trapping, isolation, and identification of a derivative of such an intermediate in the FDTS-catalyzed reaction. The chemically modified reaction intermediate is consistent with currently proposed FDTS mechanisms that do not involve an enzymatic nucleophile, and it has never been observed during any other TSase reaction. These findings establish the timing of the methylene transfer during FDTS catalysis. The presented methodology provides an important experimental tool for further studies of FDTS, which may assist efforts directed toward the rational design of inhibitors as leads for future antibiotics.

PMID:
22295882
PMCID:
PMC3296878
DOI:
10.1021/ja2120822
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for American Chemical Society Icon for PubMed Central
Loading ...
Support Center