BACKGROUND: 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. RESULTS: The complete primary structure of the fungal beta-N-acetylhexosaminidase from Aspergillus oryzae CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the N-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate - chitobiose with a stable value of binding energy during the molecular dynamics simulation. CONCLUSION: Whereas the intracellular bacterial beta-N-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal beta-N-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and N-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys448 with Cys483 stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.

• MeSH
• Aspergillus oryzae enzymologie   MeSH
• beta-N-acetylhexosaminidasy chemie   izolace a purifikace   metabolismus   MeSH
• dimerizace MeSH
• glykosylace MeSH
• koncentrace vodíkových iontů MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• počítačová simulace * MeSH
• Ramanova spektroskopie metody   MeSH
• spektroskopie infračervená s Fourierovou transformací metody   MeSH
• stabilita enzymů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-N-acetylhexosaminidasy MeSH

Glutamate carboxypeptidase II (GCPII) is a membrane peptidase expressed in the prostate, central and peripheral nervous system, kidney, small intestine, and tumor-associated neovasculature. The GCPII form expressed in the central nervous system, termed NAALADase, is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG) yielding free glutamate in the synaptic cleft, and is implicated in various pathologic conditions associated with glutamate excitotoxicity. The prostate form of GCPII, termed prostate-specific membrane antigen (PSMA), is up-regulated in cancer and used as an effective prostate cancer marker. Little is known about the structure of this important pharmaceutical target. As a type II membrane protein, GCPII is heavily glycosylated. In this paper we show that N-glycosylation is vital for proper folding and subsequent secretion of human GCPII. Analysis of the predicted N-glycosylation sites also provides evidence that these sites are critical for GCPII carboxypeptidase activity. We confirm that all predicted N-glycosylation sites are occupied by an oligosaccharide moiety and show that glycosylation at sites distant from the putative catalytic domain is critical for the NAAG-hydrolyzing activity of GCPII calling the validity of previously described structural models of GCPII into question.

• MeSH
• antigeny povrchové chemie   genetika   metabolismus   MeSH
• buněčné linie MeSH
• exprese genu MeSH
• glutamátkarboxypeptidasa II chemie   genetika   metabolismus   MeSH
• glykosylace MeSH
• hmyz MeSH
• hydrolýza MeSH
• katalýza MeSH
• kinetika MeSH
• lidé MeSH
• mutageneze cílená genetika   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny povrchové MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH