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.