Activation of bacterial lytic polysaccharide monooxygenases with cellobiose dehydrogenase
Activation of bacterial lytic polysaccharide monooxygenases with cellobiose dehydrogenase
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DOI：
10.1002/pro.3043
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发表时间：
2016-09
期刊：
影响因子：
8
通讯作者：
J. S. Loose;Zarah Forsberg;D. Kracher;Stefan Scheiblbrandner;R. Ludwig;V. Eijsink;G. Vaaje-Kolstad
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作者：
J. S. Loose;Zarah Forsberg;D. Kracher;Stefan Scheiblbrandner;R. Ludwig;V. Eijsink;G. Vaaje-Kolstad
溶解性多糖单加氧酶（LPMO）代表最近添加到碳水化合物活性酶中，并被分类为辅助活性（AA）家族9，10，11和13。LPMO对于有效降解柠檬酸多糖如纤维素或几丁质至关重要。这些酶是铜依赖性的，并利用氧化还原机制来切割依赖于分子氧和外部电子供体的糖苷键。电子可以由各种来源提供，例如化合物（例如，抗坏血酸盐）或酶（例如，纤维二糖脱氢酶，CDH，来自真菌）。在这里，我们证明，真菌CDH从Myriococcum thermophilum（MtCDH），可以作为一个电子供体的细菌家族AA10 LPMO。我们表明，采用酶作为电子供体是有利的，因为这使得动力学控制的电子供应的LPMO。通过CBP 21的甲壳素氧化的速率等于通过MtCDH的共底物（乳糖）氧化的速率，验证了在LPMO催化机制中使用两个电子。此外，由于乳糖氧化与LPMO催化速率直接相关，因此涉及间接测定LPMO活性的方法。最后，通过MtCDH的CBP 21活性位点铜的单电子还原被确定为比通过LPMO的几丁质氧化快得多。总的来说，MtCDH似乎是细菌和真菌LPMO的通用电子供体，表明它们的电子转移机制是相似的。
Lytic polysaccharide monooxygenases (LPMOs) represent a recent addition to the carbohydrate‐active enzymes and are classified as auxiliary activity (AA) families 9, 10, 11, and 13. LPMOs are crucial for effective degradation of recalcitrant polysaccharides like cellulose or chitin. These enzymes are copper‐dependent and utilize a redox mechanism to cleave glycosidic bonds that is dependent on molecular oxygen and an external electron donor. The electrons can be provided by various sources, such as chemical compounds (e.g., ascorbate) or by enzymes (e.g., cellobiose dehydrogenases, CDHs, from fungi). Here, we demonstrate that a fungal CDH from Myriococcum thermophilum (MtCDH), can act as an electron donor for bacterial family AA10 LPMOs. We show that employing an enzyme as electron donor is advantageous since this enables a kinetically controlled supply of electrons to the LPMO. The rate of chitin oxidation by CBP21 was equal to that of cosubstrate (lactose) oxidation by MtCDH, verifying the usage of two electrons in the LPMO catalytic mechanism. Furthermore, since lactose oxidation correlates directly with the rate of LPMO catalysis, a method for indirect determination of LPMO activity is implicated. Finally, the one electron reduction of the CBP21 active site copper by MtCDH was determined to be substantially faster than chitin oxidation by the LPMO. Overall, MtCDH seems to be a universal electron donor for both bacterial and fungal LPMOs, indicating that their electron transfer mechanisms are similar.