CYP105A1 mediated 3-hydroxylation of glimepiride and glibenclamide using a recombinant Bacillus megaterium whole-cell catalyst.
CYP105A1 from Streptomyces griseolus belongs to a widespread family of soluble prokaryotic cytochromes P450. For in vitro studies we established an electron transfer system, consisting of the ferredoxin Etp1(fd) and the ferredoxin reductase Arh1 from the fission yeast Schizosaccharomyces pombe. We investigated the metabolism of glibenclamide and glimepiride, hypoglycemic drugs of sulfonylurea type, and determined corresponding in vitro kinetic parameters. The resulting 3-cyclohexyl-hydroxylation activity towards glibenclamide and glimepiride was demonstrated by NMR analysis. Furthermore, the main product of glibenclamide, cis-3-hydroxy-glibenclamide is identical with the phase-1-metabolite of this drug in human. The orientation of glimepiride and glibenclamide in the active site of the enzyme is shown by a computational docking model. For high scale production of sulfonylurea derivatives, we designed whole-cell biocatalysts based on Bacillus megaterium MS941. Surprisingly, the system expressing only CYP105A1 showed a similar activity towards hydroxylation of glimepiride and glibenclamide compared to the system expressing additionally the redox partners, Arh1 and Etp1(fd)(516-618), indicating that the host strain provides a functional endogenous electron transfer system.
Kleser M, Hannemann F, Hutter M, Zapp J, Bernhardt R