1EA2

Pseudoreversion of the Catalytic Activity of Y14F by the Additional Tyrosine-to-Phenylalanine Substitution(s) in the Hydrogen Bond Network of Delta-5-3-Ketosteroid Isomerase from Pheudomonas putida Biotype B

Summary for 1EA2

• Entry DOI: 10.2210/pdb1ea2/pdb
• Related: 1C7H 1DMM 1DMN 1DMQ 1E3N 1E3R 1E3V 1E97 1OPY 4TSU
• Descriptor: STEROID DELTA-ISOMERASE (2 entities in total)
• Functional Keywords: isomerase, ketosteroid isomerase
• Biological source: PSEUDOMONAS PUTIDA
• Total number of polymer chains: 1
• Total formula weight: 14532.50

Authors

Choi, G.,Ha, N.-C.,Kim, M.-S.,Hong, B.-H.,Choi, K.Y. (deposition date: 2000-11-03, release date: 2001-11-01, Last modification date: 2024-05-08)

Primary citation

Choi, G.,Ha, N.-C.,Kim, M.-S.,Hong, B.-H.,Oh, B.-H.,Choi, K.Y.
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
Biochemistry, 40:6828-, 2001

PubMed Abstract: 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.

PubMed: 11389596
DOI: 10.1021/BI002767+

Experimental method

X-RAY DIFFRACTION (1.8 Å)