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Pyranose dehydrogenase ligand promiscuity: a generalized approach to simulate monosaccharide solvation, binding, and product formation.

Graf MM, Zhixiong L, Bren U, Haltrich D, van Gunsteren WF, Oostenbrink C - PLoS Comput. Biol. (2014)

Bottom Line: The free energy difference between β- and α-anomers (ΔGβ-α) of all d-stereoisomers in water were compared to experimental values with a good agreement.The relative binding free energies (ΔΔGbind) were calculated and, where available, compared to experimental values, approximated from Km values.The results suggest that a similar approach could be applied to study promiscuity of other enzymes.

View Article: PubMed Central - PubMed

Affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.

ABSTRACT
The flavoenzyme pyranose dehydrogenase (PDH) from the litter decomposing fungus Agaricus meleagris oxidizes many different carbohydrates occurring during lignin degradation. This promiscuous substrate specificity makes PDH a promising catalyst for bioelectrochemical applications. A generalized approach to simulate all 32 possible aldohexopyranoses in the course of one or a few molecular dynamics (MD) simulations is reported. Free energy calculations according to the one-step perturbation (OSP) method revealed the solvation free energies (ΔGsolv) of all 32 aldohexopyranoses in water, which have not yet been reported in the literature. The free energy difference between β- and α-anomers (ΔGβ-α) of all d-stereoisomers in water were compared to experimental values with a good agreement. Moreover, the free-energy differences (ΔG) of the 32 stereoisomers bound to PDH in two different poses were calculated from MD simulations. The relative binding free energies (ΔΔGbind) were calculated and, where available, compared to experimental values, approximated from Km values. The agreement was very good for one of the poses, in which the sugars are positioned in the active site for oxidation at C1 or C2. Distance analysis between hydrogens of the monosaccharide and the reactive N5-atom of the flavin adenine dinucleotide (FAD) revealed that oxidation is possible at HC1 or HC2 for pose A, and at HC3 or HC4 for pose B. Experimentally detected oxidation products could be rationalized for the majority of monosaccharides by combining ΔΔGbind and a reweighted distance analysis. Furthermore, several oxidation products were predicted for sugars that have not yet been tested experimentally, directing further analyses. This study rationalizes the relationship between binding free energies and substrate promiscuity in PDH, providing novel insights for its applicability in bioelectrochemistry. The results suggest that a similar approach could be applied to study promiscuity of other enzymes.

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Modifications made to the topology of system SUG (also refer to Supplementary Data).(A) System SUGa: improper dihedrals (ID) at stereocenters C1–C5 were turned off. Numbers indicate the name of the C-atom and the ID-position within the 5-digit ID code in Tables 1 and 3. Colors are in agreement with the coloring schemes in Figs. 5–7: green (C1, ID1), yellow (C2, ID2), red (C3, ID3), blue (C4, ID4), and black (C5, ID5). (B) System SUGab: in addition to system SUGa, proper dihedral force constants (kφ) for the ring torsional dihedral angles were lowered according to the following coloring scheme: blue (two force constants from 2.09 to 0.418 kJ mol−1 and one from 5.92 to 1.05 kJ mol−1), red (one force constant from 5.92 to 1.05 kJ mol−1), and yellow (two force constants from 3.77 to 1.05 kJ mol−1). (C) System SUGabc: in addition to system SUGab, bond angle bending force constants (kθ) for the bond angles surrounding the ring atoms (C1–C5 and O) were lowered according to following coloring scheme: blue (one bond angle from 380 to 285 kJ mol−1), red (two bond angles from 320 to 285 kJ mol−1), yellow (three bond angles from 320 to 285 kJ mol−1).
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pcbi-1003995-g002: Modifications made to the topology of system SUG (also refer to Supplementary Data).(A) System SUGa: improper dihedrals (ID) at stereocenters C1–C5 were turned off. Numbers indicate the name of the C-atom and the ID-position within the 5-digit ID code in Tables 1 and 3. Colors are in agreement with the coloring schemes in Figs. 5–7: green (C1, ID1), yellow (C2, ID2), red (C3, ID3), blue (C4, ID4), and black (C5, ID5). (B) System SUGab: in addition to system SUGa, proper dihedral force constants (kφ) for the ring torsional dihedral angles were lowered according to the following coloring scheme: blue (two force constants from 2.09 to 0.418 kJ mol−1 and one from 5.92 to 1.05 kJ mol−1), red (one force constant from 5.92 to 1.05 kJ mol−1), and yellow (two force constants from 3.77 to 1.05 kJ mol−1). (C) System SUGabc: in addition to system SUGab, bond angle bending force constants (kθ) for the bond angles surrounding the ring atoms (C1–C5 and O) were lowered according to following coloring scheme: blue (one bond angle from 380 to 285 kJ mol−1), red (two bond angles from 320 to 285 kJ mol−1), yellow (three bond angles from 320 to 285 kJ mol−1).

Mentions: The structure preparations were essentially performed as reported previously [13]. In short, a preliminary version of the 1.6 Å resolution X-ray structure of PDH (PDB code 4H7U) served as starting point [26]. The covalent monoatomic oxygen species at C(4a), which is most likely an X-ray artifact, was removed. As a glycoprotein, the structure of PDH comprised covalently attached sugar moieties at surface residues Asn-75 and Asn-294. The influence of these glycosylated residues on the active site is expected to be negligible and consequently the glycan structures were removed. A PO43- ion at the surface, which is most likely a crystallization buffer artifact, was removed as well. The amino and carboxy termini were charged; all arginines, cysteines and lysines were protonated, and all aspartates and glutamates were deprotonated. In our previous study, we propose that PDH oxidizes its sugar substrate via a general base proton abstraction [13], which requires one of the two active site histidines (His-512 and His-556) being neutrally charged. The most stable protonation state fulfilling this requirement was obtained when His-512 was fully protonated and His-556 was in its neutral state (proton at Nε). The selection of the tautomeric state for the neutral His-556 was such that in the X-ray structure its deprotonated nitrogen atom pointed towards the active site. The remaining histidines were doubly protonated, except for His-103, which is covalently attached to the FAD and was protonated at Nδ. No structure of PDH comprising a monosaccharide-substrate in the active site was available at commencement of this work. Therefore, PDH and the closely related GMC oxidoreductase pyranose 2-oxidase (P2O, EC 1.1.3.10) were superimposed with an atom-positional root-mean-square deviation (RMSD) of 0.13 nm for all heavy atoms of their sugar-binding sites. Two different P2O structures were used, in which the bound sugar roughly differs in a 180° rotation around the axis going through C2 and C5 of the tetrahydropyrane ring to allow for (di)oxidations at all possible sites (C1–C4). Superposition of PDH and P2O in complex with 3-fluoro-3-deoxy-β-d-glucose (PDB: 3PL8) [37] yielded pose A (Fig. 1A), whereas pose B (Fig. 1B) was obtained by aligning PDH with P2O in complex with 2-fluoro-2-deoxy-β-d-glucose (PDB: 2IGO) [38]. After grafting the monosaccharide coordinates into PDH's active site, the fluorine of the sugar was replaced by a hydroxyl group. This procedure ultimately yielded system PDH-SUG, with the monosaccharide bound to PDH according to pose A or pose B. For simulations of sugar without PDH, the coordinates of β-d-glucose from P2O-PDB 2IGO [38] were used. For the description of the interactions with the sugar, a united atom force field was used. Chirality around CH-groups in such a force field is enforced through an improper dihedral potential energy term. In order to allow transitions between equatorial and axial positions of the attached hydroxyl groups and to sample all 32 possible monosaccharides in a single MD simulation, following changes were made to the topology of β-d-glucose, following suggestions in references [28] and [31] as indicated in Fig. 2:


Pyranose dehydrogenase ligand promiscuity: a generalized approach to simulate monosaccharide solvation, binding, and product formation.

Graf MM, Zhixiong L, Bren U, Haltrich D, van Gunsteren WF, Oostenbrink C - PLoS Comput. Biol. (2014)

Modifications made to the topology of system SUG (also refer to Supplementary Data).(A) System SUGa: improper dihedrals (ID) at stereocenters C1–C5 were turned off. Numbers indicate the name of the C-atom and the ID-position within the 5-digit ID code in Tables 1 and 3. Colors are in agreement with the coloring schemes in Figs. 5–7: green (C1, ID1), yellow (C2, ID2), red (C3, ID3), blue (C4, ID4), and black (C5, ID5). (B) System SUGab: in addition to system SUGa, proper dihedral force constants (kφ) for the ring torsional dihedral angles were lowered according to the following coloring scheme: blue (two force constants from 2.09 to 0.418 kJ mol−1 and one from 5.92 to 1.05 kJ mol−1), red (one force constant from 5.92 to 1.05 kJ mol−1), and yellow (two force constants from 3.77 to 1.05 kJ mol−1). (C) System SUGabc: in addition to system SUGab, bond angle bending force constants (kθ) for the bond angles surrounding the ring atoms (C1–C5 and O) were lowered according to following coloring scheme: blue (one bond angle from 380 to 285 kJ mol−1), red (two bond angles from 320 to 285 kJ mol−1), yellow (three bond angles from 320 to 285 kJ mol−1).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263366&req=5

pcbi-1003995-g002: Modifications made to the topology of system SUG (also refer to Supplementary Data).(A) System SUGa: improper dihedrals (ID) at stereocenters C1–C5 were turned off. Numbers indicate the name of the C-atom and the ID-position within the 5-digit ID code in Tables 1 and 3. Colors are in agreement with the coloring schemes in Figs. 5–7: green (C1, ID1), yellow (C2, ID2), red (C3, ID3), blue (C4, ID4), and black (C5, ID5). (B) System SUGab: in addition to system SUGa, proper dihedral force constants (kφ) for the ring torsional dihedral angles were lowered according to the following coloring scheme: blue (two force constants from 2.09 to 0.418 kJ mol−1 and one from 5.92 to 1.05 kJ mol−1), red (one force constant from 5.92 to 1.05 kJ mol−1), and yellow (two force constants from 3.77 to 1.05 kJ mol−1). (C) System SUGabc: in addition to system SUGab, bond angle bending force constants (kθ) for the bond angles surrounding the ring atoms (C1–C5 and O) were lowered according to following coloring scheme: blue (one bond angle from 380 to 285 kJ mol−1), red (two bond angles from 320 to 285 kJ mol−1), yellow (three bond angles from 320 to 285 kJ mol−1).
Mentions: The structure preparations were essentially performed as reported previously [13]. In short, a preliminary version of the 1.6 Å resolution X-ray structure of PDH (PDB code 4H7U) served as starting point [26]. The covalent monoatomic oxygen species at C(4a), which is most likely an X-ray artifact, was removed. As a glycoprotein, the structure of PDH comprised covalently attached sugar moieties at surface residues Asn-75 and Asn-294. The influence of these glycosylated residues on the active site is expected to be negligible and consequently the glycan structures were removed. A PO43- ion at the surface, which is most likely a crystallization buffer artifact, was removed as well. The amino and carboxy termini were charged; all arginines, cysteines and lysines were protonated, and all aspartates and glutamates were deprotonated. In our previous study, we propose that PDH oxidizes its sugar substrate via a general base proton abstraction [13], which requires one of the two active site histidines (His-512 and His-556) being neutrally charged. The most stable protonation state fulfilling this requirement was obtained when His-512 was fully protonated and His-556 was in its neutral state (proton at Nε). The selection of the tautomeric state for the neutral His-556 was such that in the X-ray structure its deprotonated nitrogen atom pointed towards the active site. The remaining histidines were doubly protonated, except for His-103, which is covalently attached to the FAD and was protonated at Nδ. No structure of PDH comprising a monosaccharide-substrate in the active site was available at commencement of this work. Therefore, PDH and the closely related GMC oxidoreductase pyranose 2-oxidase (P2O, EC 1.1.3.10) were superimposed with an atom-positional root-mean-square deviation (RMSD) of 0.13 nm for all heavy atoms of their sugar-binding sites. Two different P2O structures were used, in which the bound sugar roughly differs in a 180° rotation around the axis going through C2 and C5 of the tetrahydropyrane ring to allow for (di)oxidations at all possible sites (C1–C4). Superposition of PDH and P2O in complex with 3-fluoro-3-deoxy-β-d-glucose (PDB: 3PL8) [37] yielded pose A (Fig. 1A), whereas pose B (Fig. 1B) was obtained by aligning PDH with P2O in complex with 2-fluoro-2-deoxy-β-d-glucose (PDB: 2IGO) [38]. After grafting the monosaccharide coordinates into PDH's active site, the fluorine of the sugar was replaced by a hydroxyl group. This procedure ultimately yielded system PDH-SUG, with the monosaccharide bound to PDH according to pose A or pose B. For simulations of sugar without PDH, the coordinates of β-d-glucose from P2O-PDB 2IGO [38] were used. For the description of the interactions with the sugar, a united atom force field was used. Chirality around CH-groups in such a force field is enforced through an improper dihedral potential energy term. In order to allow transitions between equatorial and axial positions of the attached hydroxyl groups and to sample all 32 possible monosaccharides in a single MD simulation, following changes were made to the topology of β-d-glucose, following suggestions in references [28] and [31] as indicated in Fig. 2:

Bottom Line: The free energy difference between β- and α-anomers (ΔGβ-α) of all d-stereoisomers in water were compared to experimental values with a good agreement.The relative binding free energies (ΔΔGbind) were calculated and, where available, compared to experimental values, approximated from Km values.The results suggest that a similar approach could be applied to study promiscuity of other enzymes.

View Article: PubMed Central - PubMed

Affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.

ABSTRACT
The flavoenzyme pyranose dehydrogenase (PDH) from the litter decomposing fungus Agaricus meleagris oxidizes many different carbohydrates occurring during lignin degradation. This promiscuous substrate specificity makes PDH a promising catalyst for bioelectrochemical applications. A generalized approach to simulate all 32 possible aldohexopyranoses in the course of one or a few molecular dynamics (MD) simulations is reported. Free energy calculations according to the one-step perturbation (OSP) method revealed the solvation free energies (ΔGsolv) of all 32 aldohexopyranoses in water, which have not yet been reported in the literature. The free energy difference between β- and α-anomers (ΔGβ-α) of all d-stereoisomers in water were compared to experimental values with a good agreement. Moreover, the free-energy differences (ΔG) of the 32 stereoisomers bound to PDH in two different poses were calculated from MD simulations. The relative binding free energies (ΔΔGbind) were calculated and, where available, compared to experimental values, approximated from Km values. The agreement was very good for one of the poses, in which the sugars are positioned in the active site for oxidation at C1 or C2. Distance analysis between hydrogens of the monosaccharide and the reactive N5-atom of the flavin adenine dinucleotide (FAD) revealed that oxidation is possible at HC1 or HC2 for pose A, and at HC3 or HC4 for pose B. Experimentally detected oxidation products could be rationalized for the majority of monosaccharides by combining ΔΔGbind and a reweighted distance analysis. Furthermore, several oxidation products were predicted for sugars that have not yet been tested experimentally, directing further analyses. This study rationalizes the relationship between binding free energies and substrate promiscuity in PDH, providing novel insights for its applicability in bioelectrochemistry. The results suggest that a similar approach could be applied to study promiscuity of other enzymes.

Show MeSH
Related in: MedlinePlus