Molecular Modeling of a fungal hexosaminidase from T. flavus with high substrate promiscuity

Natalia Kulik¹, Kristyna Slamova², Vladimir Kren², Rudiger Ettrich¹

¹Institute of Nanobiology and Structural Biology of GCRC, Academy of Sciences of the Czech

Republic , Zamek 136, 373 33 Nove Hrady, Czech Republic, kulik@nh.cas.cz

²Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha 4, Czech Republic

Beta-N-acetylhexosamnidases (HEX) are glycoside hydrolases from the family 20 (EC 3.2.1.52). They cleave terminal glucose/galactose residue from di-/oligosaccharides by a retaining mechanism. Apart of the ability to cleave hexoses specifically, fungal HEX tolerate a variety of substrate modifications [1-3]. This feature together with the ability to hydrolase the transglycosilation reaction makes these enzymes useful in biotransformation to produce modified carbohydrates with defined structures [2-3]. A homology model of the newly sequenced fungal hexosaminidase (Hex) from T.flavus has been generated. The new T.flavus Hex has a similar topology to A.oryzae and conserved active site amino acids, participating in the electrostatic interaction with the substrate. Differences are mainly in sequence and length of loops close to the active site. The model of T.flavus Hex has been refined by energetic modeling and molecular dynamics, and we now have a stable and convincing model in our hands.

The standard substrate, P-NP-GlcNAc, which is used in in vitro enzymatic assaying, the natural substrate di-N-acetylchitobiose, as well as NAG-thiazoline were docked in the active site of T.flavus Hex and their interaction energies were compared with experimental data. Differences from earlier models of A.oryzae, exclusive features of the T.flavus, as well as implications of the docking results are discussed.

Support from the Czech Science Foundation, no P207/11/0629, is acknowledged.

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