1cqs

CRYSTAL STRUCTURE OF D103E MUTANT WITH EQUILENINEOF KSI IN PSEUDOMONAS PUTIDACRYSTAL STRUCTURE OF D103E MUTANT WITH EQUILENINEOF KSI IN PSEUDOMONAS PUTIDA

Structural highlights 1cqs is a 2 chain structure with sequence from Pseudomonas putida. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: Related: 1opy Activity: Steroid Delta-isomerase, with EC number 5.3.3.1 Resources: FirstGlance, OCA, RCSB, PDBsum Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed 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. 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 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Ketosteroid Isomerase

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