Research Article: Pyranose Dehydrogenase Ligand Promiscuity: A Generalized Approach to Simulate Monosaccharide Solvation, Binding, and Product Formation

Date Published: December 11, 2014

Publisher: Public Library of Science

Author(s): Michael M. H. Graf, Lin Zhixiong, Urban Bren, Dietmar Haltrich, Wilfred F. van Gunsteren, Chris Oostenbrink, Alexander MacKerell

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.

Partial Text: Generally, 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. Although this feature has long been recognized to be useful in other contexts, for example in the recognition of multiple antigens by the same germline antibody [2]–[4], such enzymes are often characterized by poor overall catalytic efficiencies and termed promiscuous. 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. This was corroborated by later studies that investigated the evolutionary implications of promiscuity such as the adaption of enzymes towards novel xenobiotics, e.g. halogenated compounds or antibiotics, in the course of a few decades [6], [7]. Although systematic screens for promiscuous enzyme functions are not feasible because of the vast number of possible different substrates and reactions, there are many indications and examples that promiscuity is rather the rule than the exception [6]. Especially 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]–[10]. These studies suggest that metabolic pathways are intertwined in many unexpected ways, which ultimately gives the organism a higher survival potential under changing environmental conditions. Regulation of such metabolic pathways as well as promiscuity itself at the gene-, transcript-, protein-, and localization-level and the associated reaction conditions are other thriving research areas [1], [11]. Moreover, promiscuity is often observed for close homologs in protein families and distant homologs within superfamilies [11]. Individual family members have frequently evolved from a common ancestor through gene duplication and subsequent specialization. These members share the same fold and catalytic strategy, and consequently the main activity of one family member is often found as the promiscuous activity of another family member. Nobeli and coworkers refer to this phenomenon as ‘family’ promiscuity as opposed to ‘individual’ or ‘pure’ promiscuity, which is associated to multiple functions of a single enzyme [11]. The molecular mechanisms underlying promiscuity are manifold, including post-translational modifications, multiple domains, oligomeric states, protein flexibility, partial recognition, multiple interaction sites or a single site with diverse interacting residues, allosteric interactions, flexibility as well as size and complexity of the interaction partner, chemical scaffolds, and protonation states of active site residues [6], [11], [12]. Hydrophobic interactions, diverse hydrogen bonding, flexibility, and nonpolar van der Waals interactions combined with negligible electrostatics were found to be the main driving forces for promiscuity [11], [13]–[15]. Consequently, understanding the molecular mechanisms and energetics leading to enzyme promiscuity is a valuable asset in the field of protein design and engineering as well as drug development, and therefore they have been investigated extensively [1], [16]. In view of various causes and effects involving promiscuity, it is not surprising that the definition of the term is not exact and combinations of different definitions occur [1], [6], [11], [16], [17]. In this article, the term ‘promiscuity’ is used in the context of relaxed substrate specificity [1], [18] in order to perform similar chemical reactions on related substrates [17].

In this study, we presented a generalized approach to simulate monosaccharide solvation in water, as well as binding and product formation in the enzyme PDH. Introducing changes to the monosaccharide topology according to Fig. 2 created systems SUGa, SUGab, and SUGabc, out of which system SUGab was selected as the most suitable reference state for subsequent analysis in water. This allowed for sampling of all 32 possible aldohexopyranoses in only one MD simulation of the reference compound in water or using a handful of simulations of the reference state compounds within PDH.