Mechanistic Insight into the Cleavage Site Specificity of Collagenase VhaC to the Y-G Bonds in Collagen.

M9 collagenases have various biotechnological and medical applications due to their high activity and specificity to collagen. Although the collagenolytic mechanism of M9 collagenases has been studied, the molecular basis for their cleavage site specificity remains unknown. Here, the mechanism of the Y-G bond cleavage site specificity of M9 collagenase VhaC was studied. The double-G motif (G441 and G442) and Y549 of VhaC define a narrow S1' substrate binding pocket responsible for P1' substrate binding, among which Y549 functions as the structural determinant for the specific recognition of P1' glycine, because its bulky side chain restricts the accommodation of residues with a side chain. The S1' tyrosine corresponding to Y549 in VhaC is strictly conserved among M9 collagenases, suggesting its significance in the cleavage site specificity. This study advances the applications of M9 collagenases in disease treatment and collagen oligopeptide production.