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Alternative Thymidylate Synthase: New Intermediates Suggest New Flavin Mechanism
Author(s) -
Kohen Am,
Mishanina Tatiana,
Yu Liping
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.573.13
Subject(s) - thymidylate synthase , chemistry , flavin group , stereochemistry , cofactor , nucleophile , active site , nucleotide , enzyme , biochemistry , biology , gene , fluorouracil , genetics , chemotherapy , catalysis
Flavin‐dependent, thymidylate synthase (FDTS, ThyX ) catalyzes the last crucial step in the de novo biosynthesis of thymidylate, a DNA building block. Because FDTS is absent in humans while present in several human pathogens, its inhibition is of interest from pharmaceutical standpoint. Unlike classical thymidylate synthase (TSase, ThyA ), FDTS bypasses the need for an active‐site nucleophile. 1 FDTS still has to activate the substrate for the reaction; however, the nucleophile is not an enzymatic residue as in TSase. 2 Further understanding of the chemical mechanism of FDTS would greatly facilitate rational design of mechanism‐based inhibitors as leads for new class of antibiotic drugs. We will present the trapping of several intermediates 3 that shad new light on possible mechanism for this enzyme. By using acid and base as a quenchers in a rapid‐quench experiment, we find that the first intermediate yields different trapped compounds in different quesnchers. The second intermediate, on the other hand, forms dTMP product upon base quenching, which is surprising given that the flavin cofactor is still reduced at this stage (according to flavin's UV‐vis absorbance spectrum). The recent identification of the base‐trapped intermediate and a crystal structure suggest a new path for the catalyzed methylene transfer from the co‐factor (methylene tetrahydrofolate) and the substrate dUMP. These new findings may point to a methylene transfer from its folate‐donor to the target nucleotide through N5 of the FAD cofactor, in accordance with findings from X‐ray crystallography. 4Funding NIH RO1 GM110775