1cqs
From Proteopedia
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'''CRYSTAL STRUCTURE OF D103E MUTANT WITH EQUILENINEOF KSI IN PSEUDOMONAS PUTIDA''' | '''CRYSTAL STRUCTURE OF D103E MUTANT WITH EQUILENINEOF KSI IN PSEUDOMONAS PUTIDA''' | ||
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[[Category: Oh, B H.]] | [[Category: Oh, B H.]] | ||
[[Category: Park, S.]] | [[Category: Park, S.]] | ||
| - | [[Category: | + | [[Category: Equilenin]] |
| - | [[Category: | + | [[Category: Ksi]] |
| - | [[Category: | + | [[Category: Putida lbhb]] |
| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 13:00:59 2008'' | |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | |
Revision as of 10:01, 2 May 2008
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. 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 Page seeded by OCA on Fri May 2 13:00:59 2008
Categories: Pseudomonas putida | Single protein | Steroid Delta-isomerase | Choi, G. | Choi, K Y. | Ha, N C. | Kim, D H. | Kim, S W. | Oh, B H. | Park, S. | Equilenin | Ksi | Putida lbhb
