DC Field | Value | Language |
---|---|---|
dc.contributor.author | Choi, G | ko |
dc.contributor.author | Ha, NC | ko |
dc.contributor.author | Kim, MS | ko |
dc.contributor.author | Hong, BH | ko |
dc.contributor.author | Oh, Byung-Ha | ko |
dc.contributor.author | Choi, KY | ko |
dc.date.accessioned | 2013-03-04T10:17:45Z | - |
dc.date.available | 2013-03-04T10:17:45Z | - |
dc.date.created | 2012-02-06 | - |
dc.date.created | 2012-02-06 | - |
dc.date.issued | 2001-01 | - |
dc.identifier.citation | BIOCHEMISTRY, v.40, pp.6828 - 6835 | - |
dc.identifier.issn | 0006-2960 | - |
dc.identifier.uri | http://hdl.handle.net/10203/82394 | - |
dc.description.abstract | Delta (5)-3-ketosteroid isomerase (KSI) from Pseudomonas putida Biotype B catalyzes the allylic isomerization of Delta (5)-3-ketosteroids to their conjugated Delta (4)-isomers via a dienolate intermediate. Two electrophilic catalysts, Tyr-14 and Asp-99, are involved in a hydrogen bond network that comprises Asp99 O delta2 . . .O of Wat504 . . . Tyr-14 O eta . . . Tyr-55 O eta . . . Tyr-30 O eta 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 Angstrom resolution, respectively. Comparisons of the two structures near the catalytic center indicate that the hydrogen bond between Asp-99 O delta2 and C3-O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type enzyme by the additional Y30F/Y55F mutations. The kinetic parameters of the tyrosine mutants with the additional D99N or D99L mutation also support the idea that Asp-99 contributes to catalysis more efficiently in Y14F/Y30F/Y55F than in Y14F. In contrast to the catalytic mechanism of Y14F, the C4 proton of the steroid substrate was found to be transferred to the C6 position in Y14F/Y30F/Y55F with little exchange of the substrate 4 beta -proton with a solvent deuterium based on the reaction rate in D2O. Taken together, our findings strongly suggest that the improvement in the catalytic activity of Y14F by the additional Y30F/Y55F mutations is due to the changes in the structural integrity at the catalytic site and the resulting restoration of the proton-transfer mechanism in Y14F/Y30F/Y55F. | - |
dc.language | English | - |
dc.publisher | AMER CHEMICAL SOC | - |
dc.subject | SECONDARY S96P MUTATION | - |
dc.subject | 3-OXO-DELTA(5)-STEROID ISOMERASE | - |
dc.subject | DELTA-5-3-KETOSTEROID ISOMERASE | - |
dc.subject | KETOSTEROID ISOMERASE | - |
dc.subject | TRIOSEPHOSPHATE ISOMERASE | - |
dc.subject | CRYSTAL-STRUCTURE | - |
dc.subject | SITE | - |
dc.subject | MECHANISM | - |
dc.subject | ENZYME | - |
dc.subject | RESIDUES | - |
dc.title | Pseudoreversion of the catalytic activity of Y14F by the additional substitution(s) of tyrosine with phenylalanine in the hydrogen bond network of Delta(5)-3-ketosteroid isomerase from Pseudomonas putida Biotype B | - |
dc.type | Article | - |
dc.identifier.wosid | 000169232300013 | - |
dc.identifier.scopusid | 2-s2.0-0035849571 | - |
dc.type.rims | ART | - |
dc.citation.volume | 40 | - |
dc.citation.beginningpage | 6828 | - |
dc.citation.endingpage | 6835 | - |
dc.citation.publicationname | BIOCHEMISTRY | - |
dc.identifier.doi | 10.1021/bi002767+ | - |
dc.contributor.localauthor | Oh, Byung-Ha | - |
dc.contributor.nonIdAuthor | Choi, G | - |
dc.contributor.nonIdAuthor | Ha, NC | - |
dc.contributor.nonIdAuthor | Kim, MS | - |
dc.contributor.nonIdAuthor | Hong, BH | - |
dc.contributor.nonIdAuthor | Choi, KY | - |
dc.description.isOpenAccess | N | - |
dc.type.journalArticle | Article | - |
dc.subject.keywordPlus | SECONDARY S96P MUTATION | - |
dc.subject.keywordPlus | 3-OXO-DELTA(5)-STEROID ISOMERASE | - |
dc.subject.keywordPlus | DELTA-5-3-KETOSTEROID ISOMERASE | - |
dc.subject.keywordPlus | KETOSTEROID ISOMERASE | - |
dc.subject.keywordPlus | TRIOSEPHOSPHATE ISOMERASE | - |
dc.subject.keywordPlus | CRYSTAL-STRUCTURE | - |
dc.subject.keywordPlus | SITE | - |
dc.subject.keywordPlus | MECHANISM | - |
dc.subject.keywordPlus | ENZYME | - |
dc.subject.keywordPlus | RESIDUES | - |
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