Department of Food Science and Technology, Institute of Food Technology, University of Natural Resources and Life Sciences, Vienna, Austria.
Department of Biotechnology, Institute of Bioprocess Science and Engineering, University of Natural Resources and Life Sciences, Vienna, Austria.
FEBS J. 2023 Oct;290(19):4726-4743. doi: 10.1111/febs.16885. Epub 2023 Jun 20.
The interdomain electron transfer (IET) between the catalytic flavodehydrogenase domain and the electron-transferring cytochrome domain of cellobiose dehydrogenase (CDH) plays an essential role in biocatalysis, biosensors and biofuel cells, as well as in its natural function as an auxiliary enzyme of lytic polysaccharide monooxygenase. We investigated the mobility of the cytochrome and dehydrogenase domains of CDH, which is hypothesised to limit IET in solution by small angle X-ray scattering (SAXS). CDH from Myriococcum thermophilum (syn. Crassicarpon hotsonii, syn. Thermothelomyces myriococcoides) was probed by SAXS to study the CDH mobility at different pH and in the presence of divalent cations. By comparison of the experimental SAXS data, using pair-distance distribution functions and Kratky plots, we show an increase in CDH mobility at higher pH, indicating alterations of domain mobility. To further visualise CDH movement in solution, we performed SAXS-based multistate modelling. Glycan structures present on CDH partially masked the resulting SAXS shapes, we diminished these effects by deglycosylation and studied the effect of glycoforms by modelling. The modelling shows that with increasing pH, the cytochrome domain adopts a more flexible state with significant separation from the dehydrogenase domain. On the contrary, the presence of calcium ions decreases the mobility of the cytochrome domain. Experimental SAXS data, multistate modelling and previously reported kinetic data show how pH and divalent ions impact the closed state necessary for the IET governed by the movement of the CDH cytochrome domain.
细胞二糖脱氢酶 (CDH) 的催化黄素脱氢酶结构域和电子转移细胞色素结构域之间的域间电子转移 (IET) 在生物催化、生物传感器和生物燃料电池中以及在其作为溶菌多糖单加氧酶辅助酶的天然功能中起着至关重要的作用。我们研究了 CDH 的细胞色素和脱氢酶结构域的迁移性，据推测，这种迁移性通过小角 X 射线散射 (SAXS) 限制了溶液中的 IET。通过 SAXS 探测嗜热真菌 (Myriococcum thermophilum) 的 CDH，研究了不同 pH 值和二价阳离子存在下 CDH 的迁移性。通过比较实验 SAXS 数据，使用对距离分布函数和 Kratky 图，我们表明在较高 pH 值下 CDH 的迁移性增加，表明结构域迁移性发生变化。为了进一步观察溶液中 CDH 的运动，我们进行了基于 SAXS 的多态建模。CDH 上存在的聚糖结构部分掩盖了所得 SAXS 形状，我们通过去糖基化减少了这些影响，并通过建模研究了糖型的影响。建模表明，随着 pH 值的增加，细胞色素结构域呈现出更灵活的状态，与脱氢酶结构域的分离显著。相反，钙离子的存在降低了细胞色素结构域的迁移性。实验 SAXS 数据、多态建模和先前报道的动力学数据表明 pH 值和二价离子如何影响 IET 所需的闭合状态，而 IET 由 CDH 细胞色素结构域的运动控制。
