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

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

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
JournalBiochemistry
Volume39
Issue number5
DOIs
Publication statusPublished - 2000 Feb 8
Externally publishedYes

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steroid delta-isomerase
Pseudomonas putida
Hydrogen
Hydrogen bonds
Steroids
Catalysis
Equilenin
Ketosteroids
Mutagenesis
Isomerization
Protons
Site-Directed Mutagenesis
Crystal structure
Mutation

ASJC Scopus subject areas

  • Biochemistry

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

In: Biochemistry, Vol. 39, No. 5, 08.02.2000, p. 903-909.

Research output: Contribution to journalArticle

Choi, Gildon ; Ha, Nam Chul ; Kim, Suhng Wook ; Kim, Do Hyung ; Park, Songhee ; Oh, Byung Ha ; Choi, Kwan Yong. / 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. In: Biochemistry. 2000 ; Vol. 39, No. 5. pp. 903-909.
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title = "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",
abstract = "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 {\AA} resolution reveals that the distance between Tyr-14 Oη and Glu-99 Oε is ca. 4.2 {\AA}, 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 {\AA}, 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.",
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T1 - 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

AU - Choi, Gildon

AU - Ha, Nam Chul

AU - Kim, Suhng Wook

AU - Kim, Do Hyung

AU - Park, Songhee

AU - Oh, Byung Ha

AU - Choi, Kwan Yong

PY - 2000/2/8

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N2 - 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.

AB - 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.

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