Histidine 109 in peptidyl-prolyl cis-trans isomerase of Bacillus subtilis plays an important role in catalysis and in cyclosporin A binding

The cyclophilin of Bacillus subtilis has a moderate affinity to cyclosporin A (IC50: 120 nM) and low catalytic activity (k(cat)/K-m: 1.1 mu M-1 s(-1)) when compared to other ubiquitous peptidyl-prolyl cis-ti ans isomerases (PPIases). The active site residues V52, H90 and H109, which are not conserved within other peptidyl-prolyl cis-trans isomerases, were found to play an important role in cyclosporin A binding and catalytic activity. In this work we report on double mutations of these residues, which greatly improved cyclosporin A affinity and catalytic activity. The H90N/H109W mutation displayed an IC50 value of 46 nM whereas the V52M/H109F mutation exhibited over 18-fold higher catalytic activity than that detected for wild-type PPIase. The mutations H109W and H109F of the B. subtilis PPIase showed no change in cyclosporin A affinity and catalytic activity between pH 6 and 8. In contrast, wild-type PPIase (H109) showed up to 10-fold reduction below pH 7.5, both in cyclosporin A affinity and in catalytic activity. These findings clearly underline the importance of the unique H109 residue in the B. subtilis enzyme.