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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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Indicated are (i) the occurrences of the 32 stereoisomers as a function of time and (ii) the number of occurrences with a lifetime ≥1 ps for a particular stereoisomer.The left two panels are derived from the 100 ns MD simulation of system , the right two panels represent the 50 ns MD simulation of system  (pose A), which was selected as a representative example for the PDH-SUG complex.
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pcbi-1003995-g004: Indicated are (i) the occurrences of the 32 stereoisomers as a function of time and (ii) the number of occurrences with a lifetime ≥1 ps for a particular stereoisomer.The left two panels are derived from the 100 ns MD simulation of system , the right two panels represent the 50 ns MD simulation of system (pose A), which was selected as a representative example for the PDH-SUG complex.

Mentions: Fig. 4 shows (i) the occurrence of each of the 32 stereoisomers as a function of time and (ii) the number of occurrences with a lifetime ≥1 ps. The left two panels are derived from the 100 ns MD simulation of system , the right two panels represent the 50 ns MD simulation of system (pose A), which was selected as a representative example. System nicely samples all stereoisomers and indicates many transitions between the monosaccharides, leading to good statistics for subsequent analysis. System (pose A) shows significantly less sampling and transitions of the stereoisomers. Therefore, six MD simulations (systems , , ; two independent runs each) were conducted for each pose as mentioned previously. In some of the simulations of the PDH-SUG complexes, the unphysical reference state compound was observed to leave the active site. This may very well represent the proper behavior of these molecules, but unbound mixtures of PDH and SUG are (i) not expected to be relevant for real molecules binding to PDH and (ii) not part of the thermodynamic cycle to calculate the binding free energies according to equations (4) and (5). For this reason, simulations and for pose A and and for pose B were excluded from the following analyses and four independent simulations of each pose remained. For the time series of relevant distances between PDH and SUG for all simulations see Figures S1 and S2 in the supplementary material. The remaining four MD simulations for each pose were exponentially averaged according to equation (9) to calculate the free-energy differences (ΔG) of individual stereoisomers and their relative binding free energies (ΔΔGbind). According to Fig. 4, system (pose A) clearly samples l-stereoisomers (sugar code 17–32; 5th digit of improper dihedral code is 4) better than d-stereoisomers (sugar code 1–16; 5th digit of improper dihedral code is 2). This is not surprising, as the transitions of the large CH2-OH group attached at this position are sterically the most hindered (see 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)

Indicated are (i) the occurrences of the 32 stereoisomers as a function of time and (ii) the number of occurrences with a lifetime ≥1 ps for a particular stereoisomer.The left two panels are derived from the 100 ns MD simulation of system , the right two panels represent the 50 ns MD simulation of system  (pose A), which was selected as a representative example for the PDH-SUG complex.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263366&req=5

pcbi-1003995-g004: Indicated are (i) the occurrences of the 32 stereoisomers as a function of time and (ii) the number of occurrences with a lifetime ≥1 ps for a particular stereoisomer.The left two panels are derived from the 100 ns MD simulation of system , the right two panels represent the 50 ns MD simulation of system (pose A), which was selected as a representative example for the PDH-SUG complex.
Mentions: Fig. 4 shows (i) the occurrence of each of the 32 stereoisomers as a function of time and (ii) the number of occurrences with a lifetime ≥1 ps. The left two panels are derived from the 100 ns MD simulation of system , the right two panels represent the 50 ns MD simulation of system (pose A), which was selected as a representative example. System nicely samples all stereoisomers and indicates many transitions between the monosaccharides, leading to good statistics for subsequent analysis. System (pose A) shows significantly less sampling and transitions of the stereoisomers. Therefore, six MD simulations (systems , , ; two independent runs each) were conducted for each pose as mentioned previously. In some of the simulations of the PDH-SUG complexes, the unphysical reference state compound was observed to leave the active site. This may very well represent the proper behavior of these molecules, but unbound mixtures of PDH and SUG are (i) not expected to be relevant for real molecules binding to PDH and (ii) not part of the thermodynamic cycle to calculate the binding free energies according to equations (4) and (5). For this reason, simulations and for pose A and and for pose B were excluded from the following analyses and four independent simulations of each pose remained. For the time series of relevant distances between PDH and SUG for all simulations see Figures S1 and S2 in the supplementary material. The remaining four MD simulations for each pose were exponentially averaged according to equation (9) to calculate the free-energy differences (ΔG) of individual stereoisomers and their relative binding free energies (ΔΔGbind). According to Fig. 4, system (pose A) clearly samples l-stereoisomers (sugar code 17–32; 5th digit of improper dihedral code is 4) better than d-stereoisomers (sugar code 1–16; 5th digit of improper dihedral code is 2). This is not surprising, as the transitions of the large CH2-OH group attached at this position are sterically the most hindered (see 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