1CQS
CRYSTAL STRUCTURE OF D103E MUTANT WITH EQUILENINEOF KSI IN PSEUDOMONAS PUTIDA
1CQS の概要
| エントリーDOI | 10.2210/pdb1cqs/pdb |
| 関連するPDBエントリー | 1OPY |
| 分子名称 | PROTEIN : KETOSTEROID ISOMERASE, EQUILENIN (3 entities in total) |
| 機能のキーワード | ksi, equilenin, putida lbhb, isomerase |
| 由来する生物種 | Pseudomonas putida |
| タンパク質・核酸の鎖数 | 2 |
| 化学式量合計 | 29655.75 |
| 構造登録者 | |
| 主引用文献 | Choi, G.,Ha, N.C.,Kim, S.W.,Kim, D.H.,Park, S.,Oh, B.H.,Choi, K.Y. Asp-99 donates a hydrogen bond not to Tyr-14 but to the steroid directly in the catalytic mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B Biochemistry, 39:903-909, 2000 Cited by PubMed Abstract: Delta 5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Delta 5-3-ketosteroids at a rate approaching the diffusion limit by an intramolecular transfer of a proton. Despite the extensive studies on the catalytic mechanism, it still remains controversial whether the catalytic residue Asp-99 donates a hydrogen bond to the steroid or to Tyr-14. To clarify the role of Asp-99 in the catalysis, two single mutants of D99E and D99L and three double mutants of Y14F/D99E, Y14F/D99N, and Y14F/D99L have been prepared by site-directed mutagenesis. The D99E mutant whose side chain at position 99 is longer by an additional methylene group exhibits nearly the same kcat as the wild-type while the D99L mutant exhibits ca. 125-fold lower kcat than that of the wild-type. The mutations made at positions 14 and 99 exert synergistic or partially additive effect on kcat in the double mutants, which is inconsistent with the mechanism based on the hydrogen-bonded catalytic dyad, Asp-99 COOH...Tyr-14 OH...C3-O of the steroid. The crystal structure of D99E/D38N complexed with equilenin, an intermediate analogue, at 1.9 A resolution reveals that the distance between Tyr-14 O eta and Glu-99 O epsilon is ca. 4.2 A, which is beyond the range for a hydrogen bond, and that the distance between Glu-99 O epsilon and C3-O of the steroid is maintained to be ca. 2.4 A, short enough for a hydrogen bond to be formed. Taken together, these results strongly support the idea that Asp-99 contributes to the catalysis by donating a hydrogen bond directly to the intermediate. PubMed: 10653633DOI: 10.1021/bi991579k 主引用文献が同じPDBエントリー |
| 実験手法 | X-RAY DIFFRACTION (1.9 Å) |
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