Origin of the different pH activity profile in two homologous ketosteroid isomerases

Young Sung Yun, Tae Hee Lee, Gyu Hyun Nam, Do Soo Jang, Sejeong Shin, Byung Ha Oh, Kwan Yong Choi

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Two homologous Δ5-3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not. Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT. To investigate the role of Trp-116, we prepared the F116W mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic parameters of those mutants at different pH levels. PI-W116F exhibited significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the kcat\Km value by 2.5- to 4.7-fold compared with TI-WT at pH 3.8. The crystal structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT. The present results demonstrate that the activities of both PI-WT and TI-F116W at low pH were maintained by a tryptophan, which was able not only to lower the pKα value of the catalytic base but also to increase the substrate affinity. This is one example of the strategy nature can adopt to evolve the diversity of the catalytic function in the enzymes. Our results provide insight into deciphering the molecular evolution of the enzyme and creating novel enzymes by protein engineering.

Original languageEnglish (US)
Pages (from-to)28229-28236
Number of pages8
JournalJournal of Biological Chemistry
Issue number30
StatePublished - Jul 25 2003
Externally publishedYes


Dive into the research topics of 'Origin of the different pH activity profile in two homologous ketosteroid isomerases'. Together they form a unique fingerprint.

Cite this