β-Hexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of β-1,4-linkedN-acetylhexosamine residues from oligosaccharides and their conjugates. Heritable deficiency of this enzyme results in various forms of GalNAc-β(1,4)-[N-acetylneuraminic acid (2,3)]-Gal-β(1,4)-Glc-ceramide gangliosidosis, including Tay-Sachs disease. We have determined the x-ray crystal structure of a β-hexosaminidase from Streptomyces plicatus to 2.2 Å resolution (Protein Data Bank code 1HP4). β-Hexosaminidases are believed to use a substrate-assisted catalytic mechanism that generates a cyclic oxazolinium ion intermediate. We have solved and refined a complex between the cyclic intermediate analogueN-acetylglucosamine-thiazoline and β-hexosaminidase fromS. plicatus to 2.1 Å resolution (Protein Data Bank code1HP5). Difference Fourier analysis revealed the pyranose ring ofN-acetylglucosamine-thiazoline bound in the enzyme active site with a conformation close to that of a ⁴C₁chair. A tryptophan-lined hydrophobic pocket envelops the thiazoline ring, protecting it from solvolysis at the iminium ion carbon. Within this pocket, Tyr³⁹³ and Asp³¹³ appear important for positioning the 2-acetamido group of the substrate for nucleophilic attack at the anomeric center and for dispersing the positive charge distributed into the oxazolinium ring upon cyclization. This complex provides decisive structural evidence for substrate-assisted catalysis and the formation of a covalent, cyclic intermediate in family 20 β-hexosaminidases.

Hyaluronan (HA), a large glycosaminoglycan abundant in the extracellular matrix, is important in cell migration during embryonic development, cellular proliferation, and differentiation and has a structural role in connective tissues. The turnover of HA requires endoglycosidic breakdown by lysosomal hyaluronidase, and a congenital deficiency of hyaluronidase has been thought to be incompatible with life. However, a patient with a deficiency of serum hyaluronidase, now designated as mucopolysaccharidosis IX, was recently described. This patient had a surprisingly mild clinical phenotype, including notable periarticular soft tissue masses, mild short stature, an absence of neurological or visceral involvement, and histological and ultrastructural evidence of a lysosomal storage disease. To determine the molecular basis of mucopolysaccharidosis IX, we analyzed two candidate genes tandemly distributed on human chromosome 3p21.3 and encoding proteins with homology to a sperm protein with hyaluronidase activity. These genes, HYAL1 and HYAL2 , encode two distinct lysosomal hyaluronidases with different substrate specificities. We identified two mutations in the HYAL1 alleles of the patient, a 1412G → A mutation that introduces a nonconservative amino acid substitution (Glu268Lys) in a putative active site residue and a complex intragenic rearrangement, 1361del37ins14, that results in a premature termination codon. We further show that these two hyaluronidase genes, as well as a third recently discovered adjacent hyaluronidase gene, HYAL3 , have markedly different tissue expression patterns, consistent with differing roles in HA metabolism. These data provide an explanation for the unexpectedly mild phenotype in mucopolysaccharidosis IX and predict the existence of other hyaluronidase deficiency disorders.