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Title
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Mechanistic studies of a flavin-dependent thymidylate synthase.
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Authors
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N.Agrawal,
S.A.Lesley,
P.Kuhn,
A.Kohen.
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Ref.
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Biochemistry, 2004,
43,
10295-10301.
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PubMed id
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Abstract
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The ThyA gene that encodes for thymidylate synthase (TS) is absent in the
genomes of a large number of bacteria, including several human pathogens. Many
of these bacteria also lack the genes for dihydrofolate reductase (DHFR) and
thymidine kinase and are totally dependent on an alternative enzyme for
thymidylate synthesis. Thy1 encodes flavin-dependent TS (FDTS, previously
denoted as TSCP) and shares no sequence homology with classical TS genes.
Mechanistic studies of a FDTS from Thermotoga maritima (TM0449) are presented
here. Several isotopic labeling experiments reveal details of the catalyzed
reaction, and a chemical mechanism that is consistent with the experimental data
is proposed. The reaction proceeds via a ping-pong mechanism where nicotinamide
binding and release precedes the oxidative half-reaction. The enzyme is
primarily pro-R specific with regard to the nicotinamide (NADPH), the oxidation
of which is the rate-limiting step of the whole catalytic cascade. An
enzyme-bound flavin is reduced with an isotope effect of 25 (consistent with
H-tunneling) and exchanges protons with the solvent prior to the reduction of an
intermediate methylene. A quantitative assay was developed, and the kinetic
parameters were measured. A significant NADPH substrate inhibition and large
K(M) rationalized the slow activity reported for this enzyme in the past. These
and other findings are compared with classical TS (ThyA) catalysis in terms of
kinetic and molecular mechanisms. The differences between the FDTS proposed
mechanism and that of the classical TS are striking and invoke the notion that
mechanism-based drugs will selectively inhibit FDTS and will not have much
effect on human (and other eukaryotes) TS. Since TS activity is essential to DNA
replication, the unique mechanism of FDTS makes it an attractive target for
antibiotic drug development.
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