Fungal beta-N-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal beta-N-acetylhexosaminidase. The fungal beta-N-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from Aspergillus oryzae was purified and its sequence was determined.

beta-N-Acetylhexosaminidases (EC 3.2.1.52) belong to an enzyme family that hydrolyzes terminal beta-d-N-glucosamine and beta-d-N-galactosamine residues from oligosaccharides. In this report, we purified a novel beta-N-acetylhexosaminidase (Pcb-NAHA1) from the marine zoanthid Palythoa caribaeorum by applying ammonium sulfate fractionation, affinity chromatography on a chitin column, followed by two rounds of size exclusion chromatography. SDS-PAGE analysis indicated a single band protein of apparent homogeneity with a molecular mass of 25kDa. The purified enzyme preferentially hydrolyzed p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylglucosamide (pNP-GlcNAc) and to a lesser extent p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylgalactosamide (pNP-GalNAc). Detailed kinetic analysis using pNP-GlcNAc resulted in a specific activity of 57.9 U/mg, a K(m) value of 0.53 mM and a V(max) value of 88.1 micromol/h/mg and k(cat) value of 0.61s(-1). Furthermore, purified Pcb-NAHA1 enzyme activity was decreased by Hg Cl(2) or maltose and stimulated in the presence of Na(2)SeO(4,) BaCl(2), MgCl(2,) chondroitin 6-sulfate, and phenylmethylsulfonylfluoride. The optimum activity of Pcb-NAHA1 was observed at pH 5.0 and elevated temperatures (45-60 degrees C). Direct sequencing of proteolytic fragments generated from Pcb-NAHA1 revealed remarkable similarities to plant chitinases, which belong to family 18, although no chitinase activity was detected with Pcb-NAHA1. We conclude that beta-N-acetylhexosaminidases, representing a type of exochitinolytic activity, and endo-chitinases share common functional domains and/or may have evolved from a common ancestor.

We report the molecular cloning and characterization of two novel beta-N-acetylhexosaminidases (beta-HEX, EC 3.2.1.52) from Paenibacillus sp. strain TS12. The two beta-HEXs (Hex1 and Hex2) were 70% identical in primary structure, and the N-terminal region of both enzymes showed significant similarity with beta-HEXs belonging to glycoside hydrolase family 20 (GH20). Interestingly, however, the C-terminal region of Hex1 and Hex2 shared no sequence similarity with the GH20 beta-HEXs or other known proteins. Both recombinant enzymes, expressed in Escherichia coli BL21(DE3), hydrolyzed the beta-N-acetylhexosamine linkage of chitooligosaccharides and glycosphingolipids such as asialo GM2 and Gb4Cer in the absence of detergent. However, the enzyme was not able to hydrolyze GM2 ganglioside in the presence or in the absence of detergent. We determined three crystal structures of Hex1; the Hex1 deletion mutant Hex1-DeltaC at a resolution of 1.8 A; Hex1-DeltaC in complex with beta-N-acetylglucosamine at 1.6 A; and Hex1-DeltaC in complex with beta-N-acetylgalactosamine at 1.9 A. We made a docking model of Hex1-DeltaC with GM2 oligosaccharide, revealing that the sialic acid residue of GM2 could hinder access of the substrate to the active site cavity. This is the first report describing the molecular cloning, characterization and X-ray structure of a procaryotic beta-HEX capable of hydrolyzing glycosphingolipids.

Late-onset GM2 gangliosidosis (LOGG) subtypes late-onset Tay-Sachs (LOTS) and Sandhoff disease (LOSD) are ultra-rare neurodegenerative lysosomal storage disorders presenting with weakness, ataxia, and neuropsychiatric symptoms. Previous studies considered LOTS and LOSD clinically indistinguishable; recent studies have challenged this. We performed a scoping review to ascertain whether imaging and clinical features may differentiate these diseases.

Beta-N-acetylhexosaminidases belonging to the glycosyl hydrolase 20 (GH20) family are involved in the removal of terminal beta-glycosidacally linked N-acetylhexosamine residues. These enzymes, widely distributed in microorganisms, animals and plants, are involved in many important physiological and pathological processes, such as cell structural integrity, energy storage, pathogen defence, viral penetration, cellular signalling, fertilization, development of carcinomas, inflammatory events and lysosomal storage diseases. Nevertheless, only limited analyses of phylogenetic relationships between GH20 genes have been performed until now.

Hyaluronidases (HYALs) are endo-beta-N-acetylhexosaminidases that depolymerize not only hyaluronan but also chondroitin sulfate (CS) at the initial step of their catabolism. Although HYAL1 hydrolyzes both CS and HA, HYAL4 is a CS-specific endoglycosidase. The substrate specificity of HYAL4 and identification of amino acid residues required for its enzymatic activity have been reported. In this study, we characterized the properties of HYAL4 including the expression levels in various tissues, cellular localization, and effects of its overexpression on intracellular CS catabolism, using cultured cells as well as mouse tissues. Hyal4 mRNA and HYAL4 protein were demonstrated to be ubiquitously expressed in various organs in the mouse. HYAL4 protein was shown to be present both on cell surfaces as well as in lysosomes of rat skeletal muscle myoblasts, L6 cells. Overexpression of HYAL4 in Chinese hamster ovary cells decreased in the total amount of CS, suggesting its involvement in the cellular catabolism of CS. In conclusion, HYAL4 may be widely distributed and play various biological roles, including the intracellular depolymerization of CS.