1cqs

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spinqualitylabelsall models 1cqs, resolution 1.90Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

CRYSTAL STRUCTURE OF D103E MUTANT WITH EQUILENINEOF KSI IN PSEUDOMONAS PUTIDA

Overview

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.

About this Structure

1CQS is a Single protein structure of sequence from Pseudomonas putida with EQU as ligand. Active as Steroid Delta-isomerase, with EC number 5.3.3.1 Full crystallographic information is available from OCA.

Reference

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., Choi G, Ha NC, Kim SW, Kim DH, Park S, Oh BH, Choi KY, Biochemistry. 2000 Feb 8;39(5):903-9. PMID:10653633

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