PDBsum entry 2af6

Transferase

PDB id: 2af6

Contents

Protein chains

(+ 2 more) 245 a.a. *

Ligands

FAD ×8

BRU ×8

GOL ×14

Metals

IOD ×10

Waters ×989

* Residue conservation analysis

PDB id:

2af6

Name:

Transferase

Title:

Crystal structure of mycobacterium tuberculosis flavin dependent thymidylate synthase (mtb thyx) in the presence of co-factor fad and substrate analog 5-bromo-2'-deoxyuridine-5'-monophosphate (brdump)

Structure:

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

Source:

Mycobacterium tuberculosis. Organism_taxid: 1773. Gene: thyx. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Tetramer (from PQS)

Resolution:

2.01Å R-factor: 0.198 R-free: 0.243

Authors:

P.Sampathkumar,S.Turley,J.E.Ulmer,H.G.Rhie,C.H.Sibley,W.G.Hol

Key ref:

P.Sampathkumar et al. (2005). Structure of the Mycobacterium tuberculosis flavin dependent thymidylate synthase (MtbThyX) at 2.0A resolution. J Mol Biol, 352, 1091-1104. PubMed id: 16139296 DOI: 10.1016/j.jmb.2005.07.071

Date:

25-Jul-05 Release date: 04-Oct-05

Protein chains

P9WG57  (THYX_MYCTU) -  Flavin-dependent thymidylate synthase from Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)

Seq: 250 a.a.

Struc: 245 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

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 = BRU) matches with 95.24% similarity) + 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: 10.1016/j.jmb.2005.07.071

J Mol Biol 352:1091-1104 (2005)

PubMed id: 16139296

Structure of the Mycobacterium tuberculosis flavin dependent thymidylate synthase (MtbThyX) at 2.0A resolution.

P.Sampathkumar, S.Turley, J.E.Ulmer, H.G.Rhie, C.H.Sibley, W.G.Hol.

ABSTRACT

A novel flavin-dependent thymidylate synthase was identified recently as an essential gene in many archaebacteria and some pathogenic eubacteria. This enzyme, ThyX, is a potential antibacterial drug target, since humans and most eukaryotes lack the thyX gene and depend upon the conventional thymidylate synthase (TS) for their dTMP requirements. We have cloned and overexpressed the thyX gene (Rv2754c) from Mycobacterium tuberculosis in Escherichia coli. The M.tuberculosis ThyX (MtbThyX) enzyme complements the E.coli chi2913 strain that lacks its conventional TS activity. The crystal structure of the homotetrameric MtbThyX was determined in the presence of the cofactor FAD and the substrate analog, 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUMP). In the active site, which is formed by three monomers, FAD is bound in an extended conformation with the adenosine ring in a deep pocket and BrdUMP in a closed conformation near the isoalloxazine ring. Structure-based mutational studies have revealed a critical role played by residues Lys165 and Arg168 in ThyX activity, possibly by governing access to the carbon atom to be methylated of a totally buried substrate dUMP.

Selected figure(s)

Figure 3.
Figure 3. Stereo views of the FAD and BrdUMP-binding sites. (a) The FAD-binding site formed by residues from the E, G and H subunits. The flexible His69 from the G subunit is shown in gray; (b) the BrdUMP-binding site composed of residues from the E and H subunits. Black dashes indicate putative hydrogen bonding interactions. The important contacts between the Br and C5 of BrdUMP and N5 of the flavin ring are shown as broken red lines. Residues from subunits E, G and H are colored gold, green and cyan, respectively.

Figure 5.
Figure 5. Key residues near the substrate-binding site of MtbThyX. (a) Interactions made by distal residues Lys165 and Arg168 with the residues 107-108 from the ThyX motif, immediately lining the active site. All the residues shown are from the E subunit. Substitution of Lys165 and Arg168 by alanine abrogates the ability of MtbThyX to complement ThyA-defective E. coli (Table 4). (b) The positions of two potentially crucial tyrosine residues near the BrdUMP and FAD ligands bound to MtbThyX. The side-chain of Tyr108 prevents access to the bromine atom of BrdUMP and hence also to the C5 atom of the substrate dUMP if bound in the same manner as BrdUMP. The side-chains of Tyr108 and Tyr44 are in contact with each other as well as with bulk solvent. Our hypothesis is that alterations in the conformations of these Tyr residues enable CH[2]THF to reach the dUMP substrate bound to MtbThyX. Residues Tyr44, Tyr108, Lys165, Arg168 and the water molecule 371 are highlighted in red. Secondary structure elements from subunits E, G and H are colored gold, green and cyan, respectively.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2005, 352, 1091-1104) copyright 2005.

Figures were selected by an automated process.