 |
PDBsum entry 3g4a
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
An unusual mechanism of thymidylate biosynthesis in organisms containing the thyx gene.
|
|
|
Authors
|
|
E.M.Koehn,
T.Fleischmann,
J.A.Conrad,
B.A.Palfey,
S.A.Lesley,
I.I.Mathews,
A.Kohen.
|
|
|
Ref.
|
|
Nature, 2009,
458,
919-923.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Biosynthesis of the DNA base thymine depends on activity of the enzyme
thymidylate synthase to catalyse the methylation of the uracil moiety of
2'-deoxyuridine-5'-monophosphate. All known thymidylate synthases rely on an
active site residue of the enzyme to activate 2'-deoxyuridine-5'-monophosphate.
This functionality has been demonstrated for classical thymidylate synthases,
including human thymidylate synthase, and is instrumental in mechanism-based
inhibition of these enzymes. Here we report an example of thymidylate
biosynthesis that occurs without an enzymatic nucleophile. This unusual
biosynthetic pathway occurs in organisms containing the thyX gene, which codes
for a flavin-dependent thymidylate synthase (FDTS), and is present in several
human pathogens. Our findings indicate that the putative active site nucleophile
is not required for FDTS catalysis, and no alternative nucleophilic residues
capable of serving this function can be identified. Instead, our findings
suggest that a hydride equivalent (that is, a proton and two electrons) is
transferred from the reduced flavin cofactor directly to the uracil ring,
followed by an isomerization of the intermediate to form the product,
2'-deoxythymidine-5'-monophosphate. These observations indicate a very different
chemical cascade than that of classical thymidylate synthases or any other known
biological methylation. The findings and chemical mechanism proposed here,
together with available structural data, suggest that selective inhibition of
FDTSs, with little effect on human thymine biosynthesis, should be feasible.
Because several human pathogens depend on FDTS for DNA biosynthesis, its unique
mechanism makes it an attractive target for antibiotic drugs.
|
|
|
|
|
|
|
|
Figure 1.
Figure 1: Thymidylate synthase mechanisms. a, The chemical
mechanism for the classical thymidylate synthase catalysed
reaction^1, ^2. b, The chemical mechanism for the FDTS proposed
hitherto^11. c, The newly proposed mechanism for the FDTS that
does not rely on an enzymatic nucleophile. The conserved
enzymatic nucleophile is orange, the methylene is purple, the
reducing hydride from H[4]folate is green, and the hydride from
FADH[2] is red. R = 2'-deoxyribose-5'-phosphate; R' =
(p-aminobenzoyl)-glutamate; R'' =
adenosine-5'-pyroposphate-ribityl.
|
|
Figure 2.
Figure 2: Crystal structures of the FDTS–FAD–dUMP complex.
a, Wild-type TmFDTS (Protein Data Bank 1o26); b, S88A mutant
(Protein Data Bank 3g4a); c, S88C mutant (Protein Data Bank
3g4c). The distance between the C6 carbon of dUMP and the
reducing centre of the flavin (N5 of FAD) is 3.4 Å for all
three enzymes. The distances of the side chain of residue 88 to
C6 are 4.3, 4.5 and 4.1 Å, for wild-type FDTS, S88A and
S88C, respectively. The electron density maps are 2F[o] - F[c]
with a contour level of 1.0 sigma.
|
|
|
|
|
|
The above figures are
reprinted
from an Open Access publication published by Macmillan Publishers Ltd:
Nature
(2009,
458,
919-923)
copyright 2009.
|
|
|
Secondary reference #1
|
|
|
Title
|
|
Functional analysis of substrate and cofactor complex structures of a thymidylate synthase-Complementing protein.
|
|
|
Authors
|
|
I.I.Mathews,
A.M.Deacon,
J.M.Canaves,
D.Mcmullan,
S.A.Lesley,
S.Agarwalla,
P.Kuhn.
|
|
|
Ref.
|
|
Structure, 2003,
11,
677-690.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
Figure 2.
Figure 2. Structure of TM0449(A) View of the monomer.(B)
Topology diagram of the monomer (Westhead et al., 1998). b
strands, filled black triangles; a helices, filled blue circles.
Strand directions are indicated using upward-pointing or
downward-pointing triangles. N and C termini are labeled.(C)
View of the monomer orthogonal to the view in (A).(D) View of
the tetramer showing bound FAD molecules. Flavin ring exposed to
the surface in one of the monomers is shown with an arrow.(E)
Orthogonal view of the tetramer. The paired FAD molecules on
each side of the tetramer, cyan and black.
|
|
|
|
|
|
The above figure is
reproduced from the cited reference
with permission from Cell Press
|
|
|
Secondary reference #2
|
|
|
Title
|
|
Mechanistic studies of a flavin-Dependent thymidylate synthase.
|
|
|
Authors
|
|
N.Agrawal,
S.A.Lesley,
P.Kuhn,
A.Kohen.
|
|
|
Ref.
|
|
Biochemistry, 2004,
43,
10295-10301.
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
