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

Natallia Kulik,Vladimír Křen,Kristýna Slámová,Rüdiger Ettrich

doi:10.1186/s12859-015-0465-8

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.

Highlights

Background βN-Acetylhexosaminidases belonging to the family 20 of glycoside hydrolases (GH-20; www. cazy.org) are exo-glycosidases catalyzing the hydrolysis of terminal nonreducing β-D-GlcNAc and β-D-GalNAc units from a wide variety of glycoconjugates and playing an important role in many biological processes [1]
In hexosaminidases from A. oryzae and P. oxalicum, the loop 1 is of similar size to β-N-acetylhexosaminidase from Talaromyces flavus (TfHex), while loop 2 is shorter in the middle part (Figure 1, Additional file 1: Figures S1-S2)
In this work, the biotechnologically interesting β-N-acetylhexosaminidase from Talaromyces flavus was studied using the methods of homology modeling, molecular dynamics simulation and docking

Summary

Introduction

Background βN-Acetylhexosaminidases (hexosaminidases) belonging to the family 20 of glycoside hydrolases (GH-20; www. cazy.org) are exo-glycosidases catalyzing the hydrolysis of terminal nonreducing β-D-GlcNAc and β-D-GalNAc units from a wide variety of glycoconjugates and playing an important role in many biological processes [1]. Cazy.org) are exo-glycosidases catalyzing the hydrolysis of terminal nonreducing β-D-GlcNAc and β-D-GalNAc units from a wide variety of glycoconjugates and playing an important role in many biological processes [1]. To their primary hydrolytic activity, these enzymes have been shown to catalyze transglycosylation. The biochemical properties and structure of β-N-acetylhexosaminidase from Aspergillus oryzae as the commonly used and commercially available representative of fungal hexosaminidases has been investigated during the last few years in order to reveal the structure-activity relationships in this group of enzymes. To overcome the lack of structural information of fungal β-N-acetylhexosaminidase, a homology model of the glycosylated dimeric form of T. flavus enzyme was built, and compared to modelled fungal β-N-acetylhexosaminidases from A. oryzae [12] and Penicillium oxalicum [13], correctness of which was validated by biochemical studies and vibrational spectroscopy

Methods

Results

Conclusion