Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase

Journal Article · Thu Jun 02 00:00:00 EDT 2016 · Biochemistry

DOI:https://doi.org/10.1021/acs.biochem.6b00510· OSTI ID:1357651

Stull, Frederick W. ^[1]; Bernard, Steffen M. ^[1]; Sapra, Aparna ^[1]; Smith, Janet L. ^[1]; Zuiderweg, Erik R. P. ^[1]; Palfey, Bruce A. ^[1]

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1. Univ. of Michigan, Ann Arbor, MI (United States)

Many microorganisms use flavin-dependent thymidylate synthase (FDTS) to synthesize the essential nucleotide 2'-deoxythymidine 5'-monophosphate (dTMP) from 2'-deoxyuridine 5'-monophosphate (dUMP), 5,10-methylenetetrahydrofolate (CH₂THF), and NADPH. FDTSs have a structure that is unrelated to the thymidylate synthase used by humans and a very different mechanism. Here we report nuclear magnetic resonance evidence that FDTS ionizes N3 of dUMP using an active-site arginine. The ionized form of dUMP is largely responsible for the changes in the flavin absorbance spectrum of FDTS upon dUMP binding. dUMP analogues also suggest that the phosphate of dUMP acts as the base that removes the proton from C5 of the dUMP-methylene intermediate in the FDTS-catalyzed reaction. These findings establish additional differences between the mechanisms of FDTS and human thymidylate synthase.

• Research Organization: Argonne National Lab. (ANL), Argonne, IL (United States)
• Sponsoring Organization: National Institutes of Health (NIH); National Science Foundation (NSF)
• Grant/Contract Number: R01GM081544; GM008597; 1213620; R01DK042303; Y1-GM-1104
• OSTI ID: 1357651
• Journal Information: Biochemistry, Vol. 55, Issue 23; ISSN 0006-2960
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Flavins
Peptides and proteins
Absorption
Phosphates
Uracil