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 K m 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.
Highlights
Enzymes are perceived as being specific for both their substrates and the reactions they catalyze [1]
We propose that pyranose dehydrogenase (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
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 molecular dynamics (MD) simulation, following changes were made to the topology of b-Dglucose, following suggestions in references [28] and [31] as indicated in Fig. 2: (a) Improper dihedral angle interactions at C1–C5 were turned off (Fig. 2A) – leading to model SUGa
Summary
Enzymes are perceived as being specific for both their substrates and the reactions they catalyze [1] Deviations from such behavior are often seen as unwanted side effects or even errors in the biological function of the enzyme that come at an additional energetic cost for the organism. Starting in 1976, this paradigm started to shift when Jensen drew a link between promiscuity and protein evolution [5] He hypothesized that the first enzymes had very broad substrate specificities that evolved to more specialized forms via duplication, mutation, and selection of the corresponding genes. In the past two decades, enzyme promiscuity received considerable attention, and enzymes that can take over the function of related enzymes in an organism via their promiscuous activities have been extensively investigated [8,9,10]
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