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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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Docking of C-6 modified substrates. A. Overlay of the active sites of TfHex with docked pNP-GlcNAc (2; vivid color, yellow hydrogen bonds) and pNP-GlcNAc-sulfate (6; grey). Sodium ion in the active site with the sulfated substrate is shown. B. TfHex active site with sulfated substrate 6 in the active site. Sodium ion penetrated in the active site from water solution is shown by yellow ball. Negatively charged amino acids close to the sulfo-group are shown and labeled. Distance from Cδ atom of Glu 332 and Glu 546 to sulfur atom of substrate is 0.537-0.602 and 0.465-0.609 nm, respectively, from Cε atom of Asp 472 to sulfur atom of substrate it is 0.619-0.819 nm. C. Overlay of the active site of S. plicatus hexosaminidase with docked sulfated compound (6; vivid color) and pNP-GlcNAc (2; grey). D. Overlay of the active site of S. plicatus hexosaminidase with docked pNP-GlcNAc-uronate (5; vivid) and pNP-GlcNAc (2; grey).
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Fig8: Docking of C-6 modified substrates. A. Overlay of the active sites of TfHex with docked pNP-GlcNAc (2; vivid color, yellow hydrogen bonds) and pNP-GlcNAc-sulfate (6; grey). Sodium ion in the active site with the sulfated substrate is shown. B. TfHex active site with sulfated substrate 6 in the active site. Sodium ion penetrated in the active site from water solution is shown by yellow ball. Negatively charged amino acids close to the sulfo-group are shown and labeled. Distance from Cδ atom of Glu 332 and Glu 546 to sulfur atom of substrate is 0.537-0.602 and 0.465-0.609 nm, respectively, from Cε atom of Asp 472 to sulfur atom of substrate it is 0.619-0.819 nm. C. Overlay of the active site of S. plicatus hexosaminidase with docked sulfated compound (6; vivid color) and pNP-GlcNAc (2; grey). D. Overlay of the active site of S. plicatus hexosaminidase with docked pNP-GlcNAc-uronate (5; vivid) and pNP-GlcNAc (2; grey).

Mentions: The sulfated substrate 6 forms 5–7 hydrogen bonds with TfHex even though the interaction with Asp 472 was lost during simulations (Figure 8A). Binding of substrates with charged groups at C-6 embodied positive electrostatic energy, making more unfavorable total free energy of binding estimated by AutoDock (Table 3) and making them poor substrates. This can be explained by the presence of Glu 332, Glu 546 and Asp 472 in the vicinity of the substrate’s C-6 atom (Figure 8B). Overall, the carbohydrates with bulky substitution at C-6 position are accepted by TfHex as substrates. However, negatively charged substitutions at C-6 atom caused lower hydrolysis rates due to the less favorable binding energy and unstable interaction with the catalytic Glu residue. Additional stability of charged groups in the active site of the fungal enzyme could be maintained by small cations, such as the sodium ion, often present in the buffer (Figure 8B). On the other hand, after the consequent molecular dynamics the affinity of the bacterial hexosaminidase to charged compounds 5 and 6 is significantly lower than to product 4, corresponding well to the negligible results of the hydrolytic reactions (Tables 1 and 2).Figure 8


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)

Docking of C-6 modified substrates. A. Overlay of the active sites of TfHex with docked pNP-GlcNAc (2; vivid color, yellow hydrogen bonds) and pNP-GlcNAc-sulfate (6; grey). Sodium ion in the active site with the sulfated substrate is shown. B. TfHex active site with sulfated substrate 6 in the active site. Sodium ion penetrated in the active site from water solution is shown by yellow ball. Negatively charged amino acids close to the sulfo-group are shown and labeled. Distance from Cδ atom of Glu 332 and Glu 546 to sulfur atom of substrate is 0.537-0.602 and 0.465-0.609 nm, respectively, from Cε atom of Asp 472 to sulfur atom of substrate it is 0.619-0.819 nm. C. Overlay of the active site of S. plicatus hexosaminidase with docked sulfated compound (6; vivid color) and pNP-GlcNAc (2; grey). D. Overlay of the active site of S. plicatus hexosaminidase with docked pNP-GlcNAc-uronate (5; vivid) and pNP-GlcNAc (2; grey).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig8: Docking of C-6 modified substrates. A. Overlay of the active sites of TfHex with docked pNP-GlcNAc (2; vivid color, yellow hydrogen bonds) and pNP-GlcNAc-sulfate (6; grey). Sodium ion in the active site with the sulfated substrate is shown. B. TfHex active site with sulfated substrate 6 in the active site. Sodium ion penetrated in the active site from water solution is shown by yellow ball. Negatively charged amino acids close to the sulfo-group are shown and labeled. Distance from Cδ atom of Glu 332 and Glu 546 to sulfur atom of substrate is 0.537-0.602 and 0.465-0.609 nm, respectively, from Cε atom of Asp 472 to sulfur atom of substrate it is 0.619-0.819 nm. C. Overlay of the active site of S. plicatus hexosaminidase with docked sulfated compound (6; vivid color) and pNP-GlcNAc (2; grey). D. Overlay of the active site of S. plicatus hexosaminidase with docked pNP-GlcNAc-uronate (5; vivid) and pNP-GlcNAc (2; grey).
Mentions: The sulfated substrate 6 forms 5–7 hydrogen bonds with TfHex even though the interaction with Asp 472 was lost during simulations (Figure 8A). Binding of substrates with charged groups at C-6 embodied positive electrostatic energy, making more unfavorable total free energy of binding estimated by AutoDock (Table 3) and making them poor substrates. This can be explained by the presence of Glu 332, Glu 546 and Asp 472 in the vicinity of the substrate’s C-6 atom (Figure 8B). Overall, the carbohydrates with bulky substitution at C-6 position are accepted by TfHex as substrates. However, negatively charged substitutions at C-6 atom caused lower hydrolysis rates due to the less favorable binding energy and unstable interaction with the catalytic Glu residue. Additional stability of charged groups in the active site of the fungal enzyme could be maintained by small cations, such as the sodium ion, often present in the buffer (Figure 8B). On the other hand, after the consequent molecular dynamics the affinity of the bacterial hexosaminidase to charged compounds 5 and 6 is significantly lower than to product 4, corresponding well to the negligible results of the hydrolytic reactions (Tables 1 and 2).Figure 8

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