PDBsum entry 3g4c

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Transferase PDB id
Protein chains
209 a.a. *
FAD ×4
UMP ×4
Waters ×167
* Residue conservation analysis
PDB id:
Name: Transferase
Title: Flavine dependant thymidylate syntahse s88c mutant
Structure: Thymidylate synthase thyx. Chain: a, b, c, d. Synonym: ts, tsase. Engineered: yes. Mutation: yes
Source: Thermotoga maritima. Organism_taxid: 2336. Gene: thy1, thyx, tm_0449. Expressed in: escherichia coli. Expression_system_taxid: 562.
2.05Å     R-factor:   0.202     R-free:   0.254
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
03-Feb-09     Release date:   14-Apr-09    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
Q9WYT0  (THYX_THEMA) -  Thymidylate synthase ThyX
220 a.a.
209 a.a.*
Key:    PfamA domain  Secondary structure
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.  - Thymidylate synthase (FAD).
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: 5,10-methylenetetrahydrofolate + dUMP + NADPH = dTMP + tetrahydrofolate + NADP+
Bound ligand (Het Group name = UMP)
corresponds exactly
Bound ligand (Het Group name = FAD)
matches with 71.19% similarity
= dTMP
+ tetrahydrofolate
+ NADP(+)
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     methylation   3 terms 
  Biochemical function     transferase activity     4 terms  


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.
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.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21368151 F.Yan, and D.G.Fujimori (2011).
RNA methylation by Radical SAM enzymes RlmN and Cfr proceeds via methylene transfer and hydride shift.
  Proc Natl Acad Sci U S A, 108, 3930-3934.  
  20944226 A.E.Speers, and B.F.Cravatt (2010).
Ligands in crystal structures that aid in functional characterization.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 66, 1306-1308.  
20716366 D.Weekes, S.S.Krishna, C.Bakolitsa, I.A.Wilson, A.Godzik, and J.Wooley (2010).
TOPSAN: a collaborative annotation environment for structural genomics.
  BMC Bioinformatics, 11, 426.  
19643076 E.M.Koehn, and A.Kohen (2010).
Flavin-dependent thymidylate synthase: a novel pathway towards thymine.
  Arch Biochem Biophys, 493, 96.  
19816891 E.J.Loveridge, G.Maglia, and R.K.Allemann (2009).
The role of arginine 28 in catalysis by dihydrofolate reductase from the hyperthermophile Thermotoga maritima.
  Chembiochem, 10, 2624-2627.  
19370021 M.P.Costi, and S.Ferrari (2009).
Biochemistry: Anchors away.
  Nature, 458, 840-841.  
19762644 Y.Zhang, I.Thiele, D.Weekes, Z.Li, L.Jaroszewski, K.Ginalski, A.M.Deacon, J.Wooley, S.A.Lesley, I.A.Wilson, B.Palsson, A.Osterman, and A.Godzik (2009).
Three-dimensional structural view of the central metabolic network of Thermotoga maritima.
  Science, 325, 1544-1549.  
19459936 Z.Wang, A.Chernyshev, E.M.Koehn, T.D.Manuel, S.A.Lesley, and A.Kohen (2009).
Oxidase activity of a flavin-dependent thymidylate synthase.
  FEBS J, 276, 2801-2810.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time.