Structural Insights into the Catalytic Activity of Cyclobacterium marinum N-Acetylglucosamine Deacetylase.

Abstract

N-Acetylglucosamine deacetylase from Cyclobacterium marinum (CmCBDA) is a highly effective and selective biocatalyst for the production of d-glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). However, the underlying catalytic mechanism remains elusive. Here, we show that CmCBDA is a metalloenzyme with a preference for Ni2+ over Mn2+. Crystal structures of CmCBDA in complex with Ni2+ and Mn2+ revealed slight remodeling of the CmCBDA active site by the metal ions. We also demonstrate that CmCBDA exists as a mixture of homodimers and monomers in solution, and dimerization is indispensable for catalytic activity. A mutagenesis analysis also indicated that the active site residues Asp22, His72, and His143 as well as the residues involved in dimerization, Pro52, Trp53, and Tyr55, are essential for catalytic activity. Furthermore, a mutation on the protein surface, Lys219Glu, resulted in a 2.3-fold improvement in the deacetylation activity toward GlcNAc. Mechanistic insights obtained here may facilitate the development of CmCBDA variants with higher activities.