The conserved cis-Pro(39) residue plays a crucial role in the proper positioning of the catalytic base Asp(38) in ketosteroid isomerase from Comamonas testosteroni

Abstract

KSI (ketosteroid isomerase) from Comamonas testosteroni is a homodimeric enzyme that catalyses the allylic isomerization of Delta(5)-3-ketosteroids to their conjugated Delta(4)-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-Pro(39) residue was proposed to be involved in the proper positioning of Asp(38), a critical catalytic residue, since the residue was found not only to be structurally associated with Asp(38), but also to confer a structural rigidity on the local active-site geometry consisting of Asp(38), Pro(39), Val(40), Gly(41) and Ser(42) at the flexible loop between beta-strands B1 and B2. In order to investigate the structural role of the conserved cis-Pro(39) residue near the active site of KSI, Pro(39) 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 DeltaG(U)(H2O) (the free-energy change for unfolding in the absence of urea at 25.00 +/- 0.02 degreesC) decreased by 31.0 and 35.6 kJ/moI (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 Angstrom (0.23 nm) resolution revealed that the P39A mutation significantly disrupted the proper orientations of both d-equilenin and Asp(38), as well as the local active-site geometry near Asp(38), 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-Pro(39) residue plays a crucial role in the proper positioning of the critical catalytic base Asp(38) and in the structural integrity of the active site in KSI.