Computational study of β-N-acetylhexosaminidase from Talaromyces flavus, a glycosidase with high substrate flexibility

Abstract

Backgroundβ-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest.ResultsComputational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model.ConclusionsThe main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0465-8) contains supplementary material, which is available to authorized users.

Synthesis of Derivatized Chitooligomers using Transglycosidases Engineered from the Fungal GH20 β‐N‐Acetylhexosaminidase

“…Based on a recently published computational model of TfHex,32 the active site’s Tyr470 was selected as the promising amino acid residue to be mutated to regulate hydrolytic activity of GH20 enzymes. Mutation for Phe, His and Asn indeed brought the apparent knock‐out of the original hydrolytic activity and increased transglycosylation with substrates in the natural β‐configuration.…”

Crystal structure of native β‐N‐acetylhexosaminidase isolated from Aspergillus oryzae sheds light onto its substrate specificity, high stability, and regulation by propeptide

“…They are used in synthesis of new oligosaccharides by means of transglycosylation reactions in which a carbohydrate moiety is transferred from an activated saccharide donor to its acceptor, typically an alcohol or a carbohydrate. In particular, HEX orthologs of GH families 20 or 84 obtained from filamentous fungi, mainly from Aspergillus, Penicillium, and Talaromyces genera, proved efficient in such synthetic reactions, making fungal HEX versatile tools for preparation of novel oligosaccharides [1,[13][14][15]. Moreover, HEX have shown broad substrate specificity, tolerating a variety of unnatural substrates including carboxylates, sulfates, acyls, azides, and 4-deoxyglycosides [1].…”

“…In addition, the structures of the a and b chains of human hexosaminidases A and B have been determined [24][25][26][27]. In the absence of an experimentally determined AoHEX structure, a homology modeling approach was used previously to model AoHEX [31] and other fungal HEX [14,32]. In the absence of an experimentally determined AoHEX structure, a homology modeling approach was used previously to model AoHEX [31] and other fungal HEX [14,32].…”

“…Moreover, HEX have shown broad substrate specificity, tolerating a variety of unnatural substrates including carboxylates, sulfates, acyls, azides, and 4-deoxyglycosides [1]. In particular, HEX orthologs of GH families 20 or 84 obtained from filamentous fungi, mainly from Aspergillus, Penicillium, and Talaromyces genera, proved efficient in such synthetic reactions, making fungal HEX versatile tools for preparation of novel oligosaccharides [1,[13][14][15].…”

“…More importantly, the chitinolytic hexosaminidase from the Asian corn borer moth (Ostrinia furnacalis) has been intensively studied as a potential target for insecticides [28,29], and its structure was identified as a useful template for modeling of fungal hexosaminidases because it is the genetically closest homolog of AoHEX [16,30]. In the absence of an experimentally determined AoHEX structure, a homology modeling approach was used previously to model AoHEX [31] and other fungal HEX [14,32].…”