Enzymatic characterization and molecular modeling of an evolutionarily interesting fungal β-N-acetylhexosaminidase

• Published in: The FEBS journal Vol. 278; no. 14; pp. 2469 - 2484
• Main Authors: Ryšlavá, Helena, Kalendová, Alžběta, Doubnerová, Veronika, Skočdopol, Přemysl, Kumar, Vinay, Kukačka, Zdeněk, Pompach, Petr, Vaněk, Ondřej, Slámová, Kristýna, Bojarová, Pavla, Kulik, Natallia, Ettrich, Rudiger, Křen, Vladimír, Bezouška, Karel
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-07-2011
• Subjects: Amino Acid Sequence; bacteria; beta-N-acetylhexosaminidase; beta-N-Acetylhexosaminidases - chemistry; beta-N-Acetylhexosaminidases - genetics; beta-N-Acetylhexosaminidases - isolation & purification; beta-N-Acetylhexosaminidases - metabolism; Catalytic Domain; Conserved Sequence; deglycosylation; enzyme kinetics; Enzyme Precursors - chemistry; Enzyme Precursors - genetics; Enzyme Precursors - isolation & purification; Enzyme Precursors - metabolism; Enzyme Stability; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Fungal Proteins - metabolism; fungi; genes; Glycosylation; hexosaminidase; humans; Hydrogen-Ion Concentration; Kinetics; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - metabolism; Models, Molecular; molecular cloning; molecular dynamics; Molecular Dynamics Simulation; molecular modeling; molecular models; Molecular Sequence Data; molecular weight; Penicillium - enzymology; Penicillium - genetics; Penicillium oxalicum; Protein Structure, Secondary; Sequence Alignment; sequence analysis; Sequence Homology, Amino Acid; Substrate Specificity; Temperature
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2011.08173.x

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.