title: Enzymatic characterization and molecular modeling of an evolutionarily interesting fungal β-N-acetylhexosaminidase.
abstract: Fungal β-N-acetylhexosaminidases are inducible extracellular enzymes with many biotechnological applications. The enzyme from Penicillium oxalicum has unique enzymatic properties despite its close evolutionary relationship with other fungal hexosaminidases. It has high GalNAcase activity, tolerates substrates with the modified N-acyl group better and has some other unusual catalytic properties. In order to understand these features, we performed isolation, biochemical and enzymological characterization, molecular cloning and molecular modelling. The native enzyme is composed of two catalytic units (65 kDa each) and two propeptides (15 kDa each), yielding a molecular weight of 160 kDa. Enzyme deglycosylated by endoglycosidase H had comparable activity, but reduced stability. We have cloned and sequenced the gene coding for the entire hexosaminidase from P. oxalicum. Sufficient sequence identity of this hexosaminidase with the structurally solved enzymes from bacteria and humans with complete conservation of all catalytic residues allowed us to construct a molecular model of the enzyme. Results from molecular dynamics simulations and substrate docking supported the experimental kinetic and substrate specificity data and provided a molecular explanation for why the hexosaminidase from P. oxalicum is unique among the family of fungal hexosaminidases.
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University Prague, Prague, Czech Republic.
authors.forename: Helena
authors.initials: H
authors.lastname: Ryšlavá
authors.forename: Alžběta
authors.initials: A
authors.lastname: Kalendová
authors.forename: Veronika
authors.initials: V
authors.lastname: Doubnerová
authors.forename: Přemysl
authors.initials: P
authors.lastname: Skočdopol
authors.forename: Vinay
authors.initials: V
authors.lastname: Kumar
authors.forename: Zdeněk
authors.initials: Z
authors.lastname: Kukačka
authors.forename: Petr
authors.initials: P
authors.lastname: Pompach
authors.forename: Ondřej
authors.initials: O
authors.lastname: Vaněk
authors.forename: Kristýna
authors.initials: K
authors.lastname: Slámová
authors.forename: Pavla
authors.initials: P
authors.lastname: Bojarová
authors.forename: Natallia
authors.initials: N
authors.lastname: Kulik
authors.forename: Rudiger
authors.initials: R
authors.lastname: Ettrich
authors.forename: Vladimír
authors.initials: V
authors.lastname: Křen
authors.forename: Karel
authors.initials: K
authors.lastname: Bezouška
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D017124:Conserved Sequence; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D005656:Fungal Proteins; D001619:beta-N-Acetylhexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase