1DNA

D221(169)N MUTANT DOES NOT PROMOTE OPENING OF THE COFACTOR IMIDAZOLIDINE RING

1DNA の概要

• エントリーDOI: 10.2210/pdb1dna/pdb
• 分子名称: THYMIDYLATE SYNTHASE, 2'-DEOXYURIDINE 5'-MONOPHOSPHATE, 10-PROPARGYL-5,8-DIDEAZAFOLIC ACID, ... (4 entities in total)
• 機能のキーワード: transferase, active site mutant, reaction intermediate, methyltransferase
• 由来する生物種: Escherichia coli
• 細胞内の位置: Cytoplasm: P0A884
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 62688.66

構造登録者

Sage, C.R.,Michelitsch, M.D.,Finer-Moore, J.,Stroud, R.M. (登録日: 1998-06-25, 公開日: 1998-11-04, 最終更新日: 2024-10-30)

主引用文献

Sage, C.R.,Michelitsch, M.D.,Stout, T.J.,Biermann, D.,Nissen, R.,Finer-Moore, J.,Stroud, R.M.
D221 in thymidylate synthase controls conformation change, and thereby opening of the imidazolidine.
Biochemistry, 37:13893-13901, 1998

PubMed Abstract: In thymidylate synthase (TS), the invariant residue Asp-221 provides the only side chain that hydrogen bonds to the pterin ring of the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. All mutants of D221 except cysteine abolish activity. We have determined the crystal structures of two ternary complexes of the Escherichia coli mutant D221N. In a complex with dUMP and the antifolate 10-propargyl-5,8-dideazafolate (CB3717), dUMP is covalently bound to the active site cysteine, as usual. CB3717, which has no imidazolidine ring, is also bound in the usual productive orientation, but is less ordered than in wild-type complexes. The side chain of Asn-221 still hydrogen bonds to N3 of the quinazoline ring of CB3717, which must be in the enol form. In contrast, the structure of D221N with 5-fluoro-dUMP and 5,10-methylene-5,6,7, 8-tetrahydrofolate shows the cofactor bound in two partially occupied, nonproductive binding sites. In both binding modes, the cofactor has a closed imidazolidine ring and adopts the solution conformation of the unbound cofactor. In one of the binding sites, the pterin ring is turned around such that Asn-221 hydrogen bonds to the unprotonated N1 instead of the protonated N3 of the cofactor. This orientation blocks the conformational change required for forming covalent ternary complexes. Taken together, the two crystal structures suggest that the hydrogen bond between the side chain of Asp-221 and N3 of the cofactor is most critical during the early steps of cofactor binding, where it enforces the correct orientation of the pterin ring. Proper orientation of the cofactor appears to be a prerequisite for opening the imidazolidine ring prior to formation of the covalent steady-state intermediate in catalysis.

PubMed: 9753479
DOI: 10.1021/bi9810510

実験手法

X-RAY DIFFRACTION (2.2 Å)