title: Biochemical characterization of a novel β-N-acetylhexosaminidase from the insect Ostrinia furnacalis.
abstract: 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.
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian, PR China.
authors.forename: Yamin
authors.initials: Y
authors.lastname: Huo
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.forename: Mingbo
authors.initials: M
authors.lastname: Qu
authors.forename: Qi
authors.initials: Q
authors.lastname: Chen
authors.forename: Qing
authors.initials: Q
authors.lastname: Yang
mesh_terms: D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D006056:Golgi Apparatus / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology*; D016366:Open Reading Frames / Q000235:genetics; D010802:Phylogeny; D010843:Pichia; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D011134:Polysaccharides; D011994:Recombinant Proteins; D001619:beta-N-Acetylhexosaminidases