Pseudoreversion of the Catalytic Activity of Y14F by the Additional Substitution(s) of Tyrosine with Phenylalanine in the Hydrogen Bond Network of Δ5-3-Ketosteroid Isomerase from Pseudomonas putida Biotype B
| Autor: | Nam-Chul Ha, Kwan Yong Choi, Min Sung Kim, Gildon Choi, Byung-Ha Oh, Bee-Hak Hong |
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| Rok vydání: | 2001 |
| Předmět: | |
| Zdroj: | Biochemistry. 40:6828-6835 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi002767+ |
| Popis: | Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida Biotype B catalyzes the allylic isomerization of Δ5-3-ketosteroids to their conjugated Δ4-isomers via a dienolate intermediate. Two electrophilic catalysts, Tyr-14 and Asp-99, are involved in a hydrogen bond network that comprises Asp-99 Oδ2···O of Wat504···Tyr-14 Oη···Tyr-55 Oη···Tyr-30 Oη in the active site of P. putida KSI. Even though neither Tyr-30 nor Tyr-55 plays an essential role in catalysis by the KSI, the catalytic activity of Y14F could be increased ca. 26−51-fold by the additional Y30F and/or Y55F mutation in the hydrogen bond network. To identify the structural basis for the pseudoreversion in the KSI, crystal structures of Y14F and Y14F/Y30F/Y55F have been determined at 1.8 and 2.0 A resolution, respectively. Comparisons of the two structures near the catalytic center indicate that the hydrogen bond between Asp-99 Oδ2 and C3−O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type e... |
| Databáze: | OpenAIRE |
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