PDBsum entry 3g4a

Transferase

PDB id: 3g4a

Contents

Protein chains

218 a.a. *

Ligands

UMP ×4

FAD ×4

Waters ×159

* Residue conservation analysis

PDB id:

3g4a

Name:

Transferase

Title:

Crystal structure of flavine dependant thymidylate synthase s88a mutant from thermotoga maritima at 1.95 angstrom resolution

Structure:

Thymidylate synthase thyx. Chain: a, b, c, d. Synonym: ts, tsase. Engineered: yes. Mutation: yes

Source:

Thermotoga maritima msb8. Organism_taxid: 243274. Strain: msb8 / dsm 3109 / jcm 10099. Atcc: 43589. Gene: thy1, thyx, tm_0449. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.95Å R-factor: 0.187 R-free: 0.222

Authors:

I.I.Mathews,S.A.Lesley,A.Kohen

Key ref:

E.M.Koehn et al. (2009). An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene. Nature, 458, 919-923. PubMed id: 19370033 DOI: 10.1038/nature07973

Date:

03-Feb-09 Release date: 07-Apr-09

Protein chains

Q9WYT0  (THYX_THEMA) -  Flavin-dependent thymidylate synthase from Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8)

Seq: 220 a.a.

Struc: 218 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.1.1.148 - thymidylate synthase (FAD).
• Reaction: dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate + NADPH + H⁺ = dTMP + (6S)-5,6,7,8-tetrahydrofolate + NADP⁺

dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate (Bound ligand (Het Group name = UMP) corresponds exactly) + NADPH + H(+) (Bound ligand (Het Group name = FAD) matches with 71.19% similarity) = dTMP + (6S)-5,6,7,8-tetrahydrofolate + NADP(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1038/nature07973

Nature 458:919-923 (2009)

PubMed id: 19370033

An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene.

E.M.Koehn, T.Fleischmann, J.A.Conrad, B.A.Palfey, S.A.Lesley, I.I.Mathews, A.Kohen.

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.

Selected figure(s)

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.

Figures were selected by an automated process.