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

KSI (ketosteroid isomerase) from Comamonas testosteroni is a homodimeric enzyme that catalyses the allylic isomerization of Δ⁵-3- ketosteroids to their conjugated Δ⁴-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 β-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 ΔG_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.