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1ea2

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{{STRUCTURE_1ea2| PDB=1ea2 | SCENE= }}
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'''PSEUDOREVERSION OF THE CATALYTIC ACTIVITY OF Y14F BY THE ADDITIONAL TYROSIN-TO-PHENYLALANINE SUBSTITUTION(S) IN THE HYDROGEN BOND NETWORK OF DELTA-5-3-KETOSTEROID ISOMERASE FROM PHEUDOMONAS PUTIDA BIOTYPE B'''
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===PSEUDOREVERSION OF THE CATALYTIC ACTIVITY OF Y14F BY THE ADDITIONAL TYROSIN-TO-PHENYLALANINE SUBSTITUTION(S) IN THE HYDROGEN BOND NETWORK OF DELTA-5-3-KETOSTEROID ISOMERASE FROM PHEUDOMONAS PUTIDA BIOTYPE B===
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==Overview==
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Delta5-3-ketosteroid isomerase (KSI) from Pseudomonas putida Biotype B catalyzes the allylic isomerization of Delta5-3-ketosteroids to their conjugated Delta4-isomers via a dienolate intermediate. Two electrophilic catalysts, Tyr-14 and Asp-99, are involved in a hydrogen bond network that comprises Asp-99 Odelta2...O of Wat504...Tyr-14 Oeta...Tyr-55 Oeta.Tyr-30 Oeta 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 Odelta2 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 4beta-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.
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{{ABSTRACT_PUBMED_11389596}}
==About this Structure==
==About this Structure==
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[[Category: Oh, B H.]]
[[Category: Oh, B H.]]
[[Category: Ketosteroid isomerase]]
[[Category: Ketosteroid isomerase]]
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Revision as of 21:23, 30 June 2008

Image:1ea2.png

Template:STRUCTURE 1ea2

PSEUDOREVERSION OF THE CATALYTIC ACTIVITY OF Y14F BY THE ADDITIONAL TYROSIN-TO-PHENYLALANINE SUBSTITUTION(S) IN THE HYDROGEN BOND NETWORK OF DELTA-5-3-KETOSTEROID ISOMERASE FROM PHEUDOMONAS PUTIDA BIOTYPE B

Template:ABSTRACT PUBMED 11389596

About this Structure

1EA2 is a Single protein structure of sequence from Pseudomonas putida. Full crystallographic information is available from OCA.

Reference

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., Choi G, Ha NC, Kim MS, Hong BH, Oh BH, Choi KY, Biochemistry. 2001 Jun 12;40(23):6828-35. PMID:11389596

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