1KZJ

Crystal Structure of EcTS W80G/dUMP/CB3717 Complex

Summary for 1KZJ

• Entry DOI: 10.2210/pdb1kzj/pdb
• Related: 1KZI
• Descriptor: Thymidylate synthase, 2'-DEOXYURIDINE 5'-MONOPHOSPHATE, 10-PROPARGYL-5,8-DIDEAZAFOLIC ACID, ... (4 entities in total)
• Functional Keywords: enzyme substrate cofactor analog complex, transferase
• Biological source: Escherichia coli
• Cellular location: Cytoplasm: P0A884
• Total number of polymer chains: 6
• Total formula weight: 187296.94

Authors

Fritz, T.A.,Liu, L.,Finer-Moore, J.S.,Stroud, R.M. (deposition date: 2002-02-06, release date: 2002-07-03, Last modification date: 2024-11-06)

Primary citation

Fritz, T.A.,Liu, L.,Finer-Moore, J.S.,Stroud, R.M.
Tryptophan 80 and leucine 143 are critical for the hydride transfer step of thymidylate synthase by controlling active site access.
Biochemistry, 41:7021-7029, 2002

PubMed Abstract: Mutant forms of thymidylate synthase (TS) with substitutions at the conserved active site residue, Trp 80, are deficient in the hydride transfer step of the TS reaction. These mutants produce a beta-mercaptoethanol (beta-ME) adduct of the 2'-deoxyuridine-5'-monophosphate (dUMP) exocyclic methylene intermediate. Trp 80 has been proposed to assist hydride transfer by stabilizing a 5,6,7,8-tetrahydrofolate (THF) radical cation intermediate [Barrett, J. E., Lucero, C. M., and Schultz, P. G. (1999) J. Am. Chem. Soc. 121, 7965-7966.] formed after THF changes its binding from the cofactor pocket to a putative alternate site. To understand the molecular basis of hydride transfer deficiency in a mutant in which Trp 80 was changed to Gly, we determined the X-ray structures of this mutant Escherichia coli TS complexed with dUMP and the folate analogue 10-propargyl-5,8-dideazafolate (CB3717) and of the wild-type enzyme complexed with dUMP and THF. The mutant enzyme has a cavity in the active site continuous with bulk solvent. This cavity, sealed from bulk solvent in wild-type TS by Leu 143, would allow nucleophilic attack of beta-ME on the dUMP C5 exocyclic methylene. The structure of the wild-type enzyme/dUMP/THF complex shows that THF is bound in the cofactor binding pocket and is well positioned to transfer hydride to the dUMP exocyclic methylene. Together, these results suggest that THF does not reorient during hydride transfer and indicate that the role of Trp 80 may be to orient Leu 143 to shield the active site from bulk solvent and to optimally position the cofactor for hydride transfer.

PubMed: 12033935
DOI: 10.1021/bi012108c

Experimental method

X-RAY DIFFRACTION (2.6 Å)