Graf Michael M H, Zhixiong Lin, Bren Urban, Haltrich Dietmar, van Gunsteren Wilfred F, Oostenbrink Chris
Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
Laboratory of Physical Chemistry, Swiss Federal Institute of Technology, ETH, Zürich, Switzerland.
PLoS Comput Biol. 2014 Dec 11;10(12):e1003995. doi: 10.1371/journal.pcbi.1003995. eCollection 2014 Dec.
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.
来自腐叶分解真菌姬松茸的黄素酶吡喃糖脱氢酶（PDH）可氧化木质素降解过程中出现的多种不同碳水化合物。这种宽泛的底物特异性使PDH成为生物电化学应用中一种有前景的催化剂。本文报道了一种通用方法，可在一次或几次分子动力学（MD）模拟过程中模拟所有32种可能的己醛糖吡喃糖。根据一步扰动（OSP）方法进行的自由能计算揭示了所有32种己醛糖吡喃糖在水中的溶剂化自由能（ΔGsolv），这在文献中尚未报道。将所有d - 立体异构体在水中的β - 和α - 异头物之间的自由能差（ΔGβ-α）与实验值进行比较，结果吻合良好。此外，从MD模拟计算了以两种不同构象与PDH结合的32种立体异构体的自由能差（ΔG）。计算了相对结合自由能（ΔΔGbind），并在可行的情况下与从Km值近似得到的实验值进行比较。对于其中一种构象，即糖位于C1或C2处进行氧化的活性位点的构象，吻合度非常好。单糖的氢与黄素腺嘌呤二核苷酸（FAD）的反应性N5原子之间的距离分析表明，构象A在HC1或HC2处可能发生氧化，构象B在HC3或HC4处可能发生氧化。通过结合ΔΔGbind和重新加权的距离分析，对于大多数单糖，实验检测到的氧化产物可以得到合理的解释。此外，还预测了几种尚未经过实验测试的糖的氧化产物，为进一步分析指明了方向。本研究阐明了PDH中结合自由能与底物宽泛性之间的关系，为其在生物电化学中的应用提供了新的见解。结果表明，类似的方法可用于研究其他酶的宽泛性。
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