title: Oxidase activity of a flavin-dependent thymidylate synthase.
abstract: Flavin-dependent thymidylate synthases (FDTS) catalyze the production of dTMP from dUMP and N(5),N(10)-methylene-5,6,7,8-tetrahydrofolate (CH(2)H(4)folate). In contrast to human and other classical thymidylate synthases, the activity of FDTS depends on a FAD coenzyme, and its catalytic mechanism is very different. Several human pathogens rely on this recently discovered enzyme, making it an attractive target for novel antibiotics. Like many other flavoenzymes, FDTS can function as an oxidase, which catalyzes the reduction of O(2) to H(2)O(2), using reduced NADPH or other reducing agents. In this study, we exploit the oxidase activity of FDTS from Thermatoga maritima to probe the binding and release features of the substrates and products during its synthase activity. Results from steady-state and single-turnover experiments suggest a sequential kinetic mechanism of substrate binding during FDTS oxidase activity. CH(2)H(4)folate competitively inhibits the oxidase activity, which indicates that CH(2)H(4)folate and O(2) compete for the same reduced and dUMP-activated enzymatic complex (FDTS-FADH(2)-NADP(+)-dUMP). These studies imply that the binding of CH(2)H(4)folate precedes NADP(+) release during FDTS activity. The inhibition constant of CH(2)H(4)folate towards the oxidase activity was determined to be rather small (2 microm), which indicates a tight binding of CH(2)H(4)folate to the FDTS-FADH(2)-NADP(+)-dUMP complex.
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.forename: Anatoly
authors.initials: A
authors.lastname: Chernyshev
authors.forename: Eric M
authors.initials: EM
authors.lastname: Koehn
authors.forename: Tony D
authors.initials: TD
authors.lastname: Manuel
authors.forename: Scott A
authors.initials: SA
authors.lastname: Lesley
authors.forename: Amnon
authors.initials: A
authors.lastname: Kohen
mesh_terms: D005415:Flavins / Q000378:metabolism*; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D020124:Thermotoga maritima / Q000201:enzymology*; D013940:Thymidylate Synthase / Q000378:metabolism*
chemical_list: D005415:Flavins; D009249:NADP; D010088:Oxidoreductases; D013940:Thymidylate Synthase