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 (Kcat/ Km: 1.1 microM-1 s-1) when compared to other ubiquitous peptidyl-prolyl cis-trans 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.
Achenbach
T V
TV
Philipps-Universität Marburg, Germany.
Göthel
S F
SF
Marahiel
M A
MA
D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016572:Cyclosporine / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006639:Histidine / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019696:Peptidylprolyl Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D006639:Histidine; D016572:Cyclosporine; D019696:Peptidylprolyl Isomerase