Asp-99 donates a hydrogen bond not to Tyr-14 but to the steroid directly in the catalytic mechanism of Δ5-3-ketosteroid isomerase from Pseudomonas putida biotype B

Gildon Choi, Nam Chul Ha, Suhng Wook Kim, Do Hyung Kim, Songhee Park, Byung Ha Oh, Kwan Yong Choi

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38 Citations (Scopus)


Δ5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ5-3-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 k(cat) as the wild-type while the D99L mutant exhibits ca. 125-fold lower k(cat) than that of the wild-type. The mutations made at positions 14 and 99 exert synergistic or partially additive effect on k(cat) in the double mutants, which is inconsistent with the mechanism based on the hydrogen- bonded catalytic diad, 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 Å resolution reveals that the distance between Tyr-14 Oη and Glu-99 Oε is ca. 4.2 Å, which is beyond the range for a hydrogen bond, and that the distance between Glu-99 Oε and C3-O of the steroid is maintained to be ca. 2.4 Å, 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.

Original languageEnglish
Pages (from-to)903-909
Number of pages7
Issue number5
Publication statusPublished - 2000 Feb 8
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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