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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.

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Related in: MedlinePlus

Active site of hexosaminidases with dockedpNP-GlcNAc-sulfate 6. Active site of hexosaminidases with docked pNP-GlcNAc-sulfate 6 after molecular dynamics simulation with shown residues at the distance less than 0.3 nm from the sulfate group of the substrate. Loop 2 in the vicinity of the sulfate is marked by yellow dots: A. S. plicatus hexosaminidase B. TfHex.
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Fig9: Active site of hexosaminidases with dockedpNP-GlcNAc-sulfate 6. Active site of hexosaminidases with docked pNP-GlcNAc-sulfate 6 after molecular dynamics simulation with shown residues at the distance less than 0.3 nm from the sulfate group of the substrate. Loop 2 in the vicinity of the sulfate is marked by yellow dots: A. S. plicatus hexosaminidase B. TfHex.

Mentions: In the beginning of the simulation of hexosaminidase from S. plicatus the binding energy of uronate 5 was favorable, however, during the simulation the interaction with catalytic Asp 313 and Glu 314 residues was lost. In case of the sulfated substrate 6 docking into the active site of hexosaminidase from S. plicatus was successful only when applying flexibility to the amino acid residues. The induced fit shifted the positions of side chains of Arg 162, Asp 395, Glu 444 and catalytic Asp 313 and Glu 314 to accommodate the sulfo-group (Figure 8). Comparison of the conformation of the bacterial and fungal hexosaminidases in proximity of the substrate C-6 atoms revealed the larger size of the fungal binding pocket as a consequence of longer loop 2 (Figure 9).Figure 9


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)

Active site of hexosaminidases with dockedpNP-GlcNAc-sulfate 6. Active site of hexosaminidases with docked pNP-GlcNAc-sulfate 6 after molecular dynamics simulation with shown residues at the distance less than 0.3 nm from the sulfate group of the substrate. Loop 2 in the vicinity of the sulfate is marked by yellow dots: A. S. plicatus hexosaminidase B. TfHex.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig9: Active site of hexosaminidases with dockedpNP-GlcNAc-sulfate 6. Active site of hexosaminidases with docked pNP-GlcNAc-sulfate 6 after molecular dynamics simulation with shown residues at the distance less than 0.3 nm from the sulfate group of the substrate. Loop 2 in the vicinity of the sulfate is marked by yellow dots: A. S. plicatus hexosaminidase B. TfHex.
Mentions: In the beginning of the simulation of hexosaminidase from S. plicatus the binding energy of uronate 5 was favorable, however, during the simulation the interaction with catalytic Asp 313 and Glu 314 residues was lost. In case of the sulfated substrate 6 docking into the active site of hexosaminidase from S. plicatus was successful only when applying flexibility to the amino acid residues. The induced fit shifted the positions of side chains of Arg 162, Asp 395, Glu 444 and catalytic Asp 313 and Glu 314 to accommodate the sulfo-group (Figure 8). Comparison of the conformation of the bacterial and fungal hexosaminidases in proximity of the substrate C-6 atoms revealed the larger size of the fungal binding pocket as a consequence of longer loop 2 (Figure 9).Figure 9

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
Related in: MedlinePlus