Origin of the different pH activity profile in two homologous ketosteroid isomerases.
Two homologous Delta5-3-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 pKa 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.
Yun
Young Sung
YS
Division of Molecular and Life Sciences, the National Research Laboratory of Protein Folding and Engineering, Pohang University of Science and Technology, Pohang 790-784, South Korea.
Lee
Tae-Hee
TH
Nam
Gyu Hyun
GH
Jang
Do Soo
DS
Shin
Sejeong
S
Oh
Byung-Ha
BH
Choi
Kwan Yong
KY
D001224:Aspartic Acid; D002942:Circular Dichroism; D020604:Comamonas testosteroni / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D006863:Hydrogen-Ion Concentration; D007664:Ketosteroids / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D016958:Pseudomonas putida / Q000201:enzymology*; D013229:Stearic Acids; D013251:Steroid Isomerases / Q000737:chemistry* / Q000378:metabolism
C403718:10-ketostearic acid; D007664:Ketosteroids; D013229:Stearic Acids; D001224:Aspartic Acid; D010649:Phenylalanine; D013251:Steroid Isomerases; D010100:Oxygen