Biochemical characterization of a novel β-N-acetylhexosaminidase from the insect Ostrinia furnacalis.

The β-N-acetylhexosaminidase FDL specifically removes the β-1,2-GlcNAc residue conjugated to the α-1,3-mannose residue of the core structure of insect N-glycans, playing significant physiological roles in post-translational modification in the Golgi apparatus. Little is known about its enzymatic properties. We obtained the OfFDL gene from the insect Ostrinia furnacalis by RT-PCR. The full length cDNA of FDL is 2241 bp carrying an opening reading frame of 1923 bp encoding 640 amino acids. The recombinant protein OfFDL in a soluble and active form was obtained with high purity through a two-step purification strategy. The recombinant OfFDL exclusively hydrolyzes the terminal β-1,2-GlcNAc residue from the α-1,3 branch instead of the α-1,6 branch of the substrate GnGn-PA. Several kinetic parameters including kcat/Km values toward four artificial substrates and Ki values of three representative hexosaminidase inhibitors were obtained.