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.
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.
authors.affiliation: Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, South Korea.
authors.forename: G
authors.initials: G
authors.lastname: Choi
authors.forename: N C
authors.initials: NC
authors.lastname: Ha
authors.forename: M S
authors.initials: MS
authors.lastname: Kim
authors.forename: B H
authors.initials: BH
authors.lastname: Hong
authors.forename: B H
authors.initials: BH
authors.lastname: Oh
authors.forename: K Y
authors.initials: KY
authors.lastname: Choi
mesh_terms: D020106:Acrylamide; D019943:Amino Acid Substitution / Q000235:genetics*; D000735:Androstenedione / Q000737:chemistry; D002384:Catalysis; D018360:Crystallography, X-Ray; D017666:Deuterium Oxide / Q000737:chemistry; D004789:Enzyme Activation / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry* / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013251:Steroid Isomerases / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
chemical_list: D020106:Acrylamide; D000735:Androstenedione; D014443:Tyrosine; D010649:Phenylalanine; C003528:5-androstene-3,17-dione; D014364:Tryptophan; D013251:Steroid Isomerases; C022660:steroid delta-isomerase; D017666:Deuterium Oxide