Insights into the catalytic mechanism of Grimontia hollisae collagenase through structural and mutational analyses

Abstract

Grimontia hollisae collagenase (Ghcol) exhibits high collagen‐degrading activity. To explore its catalytic mechanism, its substrate (Gly‐Pro‐Hyp‐Gly‐Pro‐Hyp, GPOGPO)‐complexed crystal structure was determined at 2.0 Å resolution. A water molecule was observed near the active‐site zinc ion. Since this water was not observed in the product (GPO)‐complexed Ghcol, it was hypothesized that the GPOGPO‐complexed Ghcol structure reflects a Michaelis complex, providing a structural basis for understanding the catalytic mechanism. Analyses of the active‐site geometry and site‐directed mutagenesis of the active‐site tyrosine residues revealed that Glu493 and Tyr564 were essential for catalysis, suggesting that Glu493 functions as an acid and base catalyst while Tyr564 stabilizes the tetrahedral complex in the transition state. These results shed light on the catalytic mechanism of bacterial collagenase.

Thermostable Bacterial Collagenolytic Proteases: A Review

“…Recently, the crystal structure of Grimontia hollisae collagenase (Ghcol) complexed with its substrate (Gly-Pro-Hyp-Gly-Pro-Hyp, GPOGPO) was determined in an attempt to understand the catalytic mechanism. Combining active-site geometry and site-directed mutagenesis, this study revealed that Glu493 and Tyr564 were essential for catalysis [ 42 ]. Moreover, a structure-based report revealed the collagenase module of Vibrio collagenase VhaC (an M9A collagenase with optimal enzymatic reaction conditions at 40°C and pH 8.0), recognizing the triple-helical collagen by its activator domain, followed by the subsequent cleavage by the peptidase domain along with the closing movement of the collagenase module.…”