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

Kulik N, Slámová K, Ettrich R, Křen V - BMC Bioinformatics (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Structure and Function of Proteins, Institute of Nanobiology and Structural Biology of GCRC, Academy of Sciences of the Czech Republic, Zamek 136, 37333, Nove Hrady, Czech Republic. kulik@nh.cas.cz.

ABSTRACT

Background: β-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.

Results: Computational 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.

Conclusions: The 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.

Show MeSH
Structures of ligands docked in the active sites of β-N-acetylhexosaminidases. Ligands are: 1 – chitobiose; 2 – pNP-GlcNAc; 3 – pNP-GalNAc; 4 – GlcNAc; 5 – pNP-GlcNAc-6-uronate; 6 – pNP-GlcNAc-6-sulfate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4384365&req=5

Fig6: Structures of ligands docked in the active sites of β-N-acetylhexosaminidases. Ligands are: 1 – chitobiose; 2 – pNP-GlcNAc; 3 – pNP-GalNAc; 4 – GlcNAc; 5 – pNP-GlcNAc-6-uronate; 6 – pNP-GlcNAc-6-sulfate.

Mentions: For the current study a set of six compounds (Figure 6) was selected for molecular dynamics simulation with hexosaminidases from the fungus Talaromyces flavus and from the bacterium Streptomyces plicatus, which is one of the first enzymes of this group that has been explored in detail and features a rather narrow substrate flexibility [14] (Table 1). The artificial substrate of β-N-acetylhexosaminidases p-nitrophenyl 2-acetamido-2-deoxy-β-D-glucosaminide (pNP-GlcNAc, 2) has been set as a standard substrate in this work and is used as a reference for the identification of binding affinity and interactions of substrates in the active sites of the enzymes. The other reported compounds are as follows (Figure 6): chitobiose (1, natural substrate of chitinolytic hexosaminidases); pNP-GalNAc (3, C-4 epimer of the standard substrate); N-acetylglucosamine (4, product of hydrolysis of 1 and 2); pNP-GlcNAc-6-uronate (5, C-6 oxidized derivative of 2) and pNP-GlcNAc-6-sulfate (6, C-6 negatively charged derivative of 2). The results of the experiments and calculation of the binding energies of equilibrated complexes are presented in Tables 1 and 2, respectively.Figure 6


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

Kulik N, Slámová K, Ettrich R, Křen V - BMC Bioinformatics (2015)

Structures of ligands docked in the active sites of β-N-acetylhexosaminidases. Ligands are: 1 – chitobiose; 2 – pNP-GlcNAc; 3 – pNP-GalNAc; 4 – GlcNAc; 5 – pNP-GlcNAc-6-uronate; 6 – pNP-GlcNAc-6-sulfate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384365&req=5

Fig6: Structures of ligands docked in the active sites of β-N-acetylhexosaminidases. Ligands are: 1 – chitobiose; 2 – pNP-GlcNAc; 3 – pNP-GalNAc; 4 – GlcNAc; 5 – pNP-GlcNAc-6-uronate; 6 – pNP-GlcNAc-6-sulfate.
Mentions: For the current study a set of six compounds (Figure 6) was selected for molecular dynamics simulation with hexosaminidases from the fungus Talaromyces flavus and from the bacterium Streptomyces plicatus, which is one of the first enzymes of this group that has been explored in detail and features a rather narrow substrate flexibility [14] (Table 1). The artificial substrate of β-N-acetylhexosaminidases p-nitrophenyl 2-acetamido-2-deoxy-β-D-glucosaminide (pNP-GlcNAc, 2) has been set as a standard substrate in this work and is used as a reference for the identification of binding affinity and interactions of substrates in the active sites of the enzymes. The other reported compounds are as follows (Figure 6): chitobiose (1, natural substrate of chitinolytic hexosaminidases); pNP-GalNAc (3, C-4 epimer of the standard substrate); N-acetylglucosamine (4, product of hydrolysis of 1 and 2); pNP-GlcNAc-6-uronate (5, C-6 oxidized derivative of 2) and pNP-GlcNAc-6-sulfate (6, C-6 negatively charged derivative of 2). The results of the experiments and calculation of the binding energies of equilibrated complexes are presented in Tables 1 and 2, respectively.Figure 6

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Structure and Function of Proteins, Institute of Nanobiology and Structural Biology of GCRC, Academy of Sciences of the Czech Republic, Zamek 136, 37333, Nove Hrady, Czech Republic. kulik@nh.cas.cz.

ABSTRACT

Background: β-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.

Results: Computational 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.

Conclusions: The 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.

Show MeSH