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Biochem. J. (2003) 375 (297–305) (Printed in Great Britain)

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The conserved cis-Pro³⁹ residue plays a crucial role in the proper positioning of the catalytic base Asp³⁸ in ketosteroid isomerase from Comamonas testosteroni

Gyu Hyun NAM*†, Sun-Shin CHA‡, Young Sung YUN*†, Yun Hee OH*†, Bee Hak HONG*†, Heung-Soo LEE‡ and Kwan Yong CHOI*†¹

*National Research Laboratory of Protein Folding and Engineering, Pohang 790-784, Republic of Korea, †Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea, and ‡Beamline Research Division, Pohang Accelerator Laboratory, Pohang 790-784, Republic of Korea

KSI (ketosteroid isomerase) from Comamonas testosteroni is a homodimeric enzyme that catalyses the allylic isomerization of D⁵-3-ketosteroids to their conjugated D⁴-isomers at a reaction rate equivalent to the diffusion-controlled limit. Based on the structural analysis of KSI at a high resolution, the conserved cis-Pro³⁹ residue was proposed to be involved in the proper positioning of Asp³⁸, a critical catalytic residue, since the residue was found not only to be structurally associated with Asp³⁸, but also to confer a structural rigidity on the local active-site geometry consisting of Asp³⁸, Pro³⁹, Val⁴⁰, Gly⁴¹ and Ser⁴² at the flexible loop between b-strands B1 and B2. In order to investigate the structural role of the conserved cis-Pro³⁹ residue near the active site of KSI, Pro³⁹ was replaced with alanine or glycine. The free energy of activation for the P39A and P39G mutants increased by 10.5 and 16.7 kJ/mol (2.5 and 4.0 kcal/mol) respectively, while DG_U^H2O (the free-energy change for unfolding in the absence of urea at 25.00±0.02 °C) decreased by 31.0 and 35.6 kJ/mol (7.4 and 8.5 kcal/mol) respectively, compared with the wild-type enzyme. The crystal structure of the P39A mutant in complex with d-equilenin [d-1,3,5(10),6,8-estrapentaen-3-ol-17-one], a reaction intermediate analogue, determined at 2.3 Å (0.23 nm) resolution revealed that the P39A mutation significantly disrupted the proper orientations of both d-equilenin and Asp³⁸, as well as the local active-site geometry near Asp³⁸, which resulted in substantial decreases in the activity and stability of KSI. Upon binding 1-anilinonaphthalene-8-sulphonic acid, the fluorescence intensities of the P39A and P39G mutants were increased drastically, with maximum wavelengths blue-shifted upon binding, indicating that the mutations might alter the hydrophobic active site of KSI. Taken together, our results demonstrate that the conserved cis-Pro³⁹ residue plays a crucial role in the proper positioning of the critical catalytic base Asp³⁸ and in the structural integrity of the active site in KSI.

Key words: active-site geometry, cis-proline, ketosteroid isomerase, site-directed mutagenesis, structural integrity.

Abbreviations used: 5-AND, 5-androstene-3,17-dione; ANS, 1-anilinonaphthalene-8-sulphonic acid; B factor, average temperature factor; d-equilenin, d-1,3,5(10),6,8-estrapentaen-3-ol-17-one; DG_U^H2O, free-energy change for unfolding in the absence of urea at 25.00±0.02 °C; KSI, ketosteroid isomerase; SH, Src homology; T_m, melting temperature.

¹To whom correspondence should be addressed (e-mail kchoi@postech.ac.kr).

The atomic coordinates for the crystal structure of P39A in complex with d-equilenin have been deposited at Brookhaven Protein Data Bank (code, 1OGZ).

Received 17 February 2003/27 June 2003; accepted 10 July 2003

Published as BJ Immediate Publication 10 July 2003, DOI 10.1042/BJ20030263

The Biochemical Society, London ©2003