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Abstract：
BACKGROUND: Oxidative enzymes targeting lignocellulosic substrates are presently classified into various auxiliary activity (AA) families within the carbohydrate-active enzyme (CAZy) database. Among these, the fungal AA3 glucose-methanol-choline (GMC) oxidoreductases with varying auxiliary activities are attractive sustainable biocatalysts and important for biological function. CAZy AA3 enzymes are further subdivided into four subfamilies, with the large AA3_2 subfamily displaying diverse substrate specificities. However, limited numbers of enzymes in the AA3_2 subfamily are currently biochemically characterized, which limits the homology-based mining of new AA3_2 oxidoreductases. Importantly, novel enzyme activities may be discovered from the uncharacterized parts of this large subfamily.
RESULTS: In this study, phylogenetic analyses employing a sequence similarity network (SSN) and maximum likelihood trees were used to cluster AA3_2 sequences. A total of 27 AA3_2 proteins representing different clusters were selected for recombinant production. Among them, seven new AA3_2 oxidoreductases were successfully produced, purified, and characterized. These enzymes included two glucose dehydrogenases (TaGdhA and McGdhA), one glucose oxidase (ApGoxA), one aryl alcohol oxidase (PsAaoA), two aryl alcohol dehydrogenases (AsAadhA and AsAadhB), and one novel oligosaccharide (gentiobiose) dehydrogenase (KiOdhA). Notably, two dehydrogenases (TaGdhA and KiOdhA) were found with the ability to utilize phenoxy radicals as an electron acceptor. Interestingly, phenoxy radicals were found to compete with molecular oxygen in aerobic environments when serving as an electron acceptor for two oxidases (ApGoxA and PsAaoA), which sheds light on their versatility. Furthermore, the molecular determinants governing their diverse enzymatic functions were discussed based on the homology model generated by AlphaFold.
CONCLUSIONS: The phylogenetic analyses and biochemical characterization of AA3_2s provide valuable guidance for future investigation of AA3_2 sequences and proteins. A clear correlation between enzymatic function and SSN clustering was observed. The discovery and biochemical characterization of these new AA3_2 oxidoreductases brings exciting prospects for biotechnological applications and broadens our understanding of their biological functions.
RESULTS: In this study, phylogenetic analyses employing a sequence similarity network (SSN) and maximum likelihood trees were used to cluster AA3_2 sequences. A total of 27 AA3_2 proteins representing different clusters were selected for recombinant production. Among them, seven new AA3_2 oxidoreductases were successfully produced, purified, and characterized. These enzymes included two glucose dehydrogenases (TaGdhA and McGdhA), one glucose oxidase (ApGoxA), one aryl alcohol oxidase (PsAaoA), two aryl alcohol dehydrogenases (AsAadhA and AsAadhB), and one novel oligosaccharide (gentiobiose) dehydrogenase (KiOdhA). Notably, two dehydrogenases (TaGdhA and KiOdhA) were found with the ability to utilize phenoxy radicals as an electron acceptor. Interestingly, phenoxy radicals were found to compete with molecular oxygen in aerobic environments when serving as an electron acceptor for two oxidases (ApGoxA and PsAaoA), which sheds light on their versatility. Furthermore, the molecular determinants governing their diverse enzymatic functions were discussed based on the homology model generated by AlphaFold.
CONCLUSIONS: The phylogenetic analyses and biochemical characterization of AA3_2s provide valuable guidance for future investigation of AA3_2 sequences and proteins. A clear correlation between enzymatic function and SSN clustering was observed. The discovery and biochemical characterization of these new AA3_2 oxidoreductases brings exciting prospects for biotechnological applications and broadens our understanding of their biological functions.
摘要：
背景:靶向木质纤维素底物的氧化酶目前在碳水化合物活性酶(CAZy)数据库内被分类为各种辅助活性(AA)家族。其中,具有不同辅助活性的真菌AA3葡萄糖-甲醇-胆碱（GMC）氧化还原酶是有吸引力的可持续生物催化剂，对生物学功能很重要。CAZyAA3酶进一步细分为四个亚家族，大的AA3_2亚族表现出不同的底物特异性。然而,AA3_2亚家族中有限数量的酶目前在生物化学上进行了表征，这限制了基于同源性的新AA3_2氧化还原酶的挖掘。重要的是,新的酶活性可以从这个大亚家族的未表征部分中发现。
结果：在这项研究中，采用序列相似性网络（SSN）和最大似然树的系统发育分析用于对AA3_2序列进行聚类。选择代表不同簇的总共27种AA3_2蛋白用于重组生产。其中,成功生产了7种新的AA3_2氧化还原酶，纯化,和特点。这些酶包括两种葡萄糖脱氢酶（TaGdhA和McGdhA），一种葡萄糖氧化酶（ApGoxA），一种芳基醇氧化酶（PsAaoA），两种芳基醇脱氢酶（AsAadhA和AsAadhB），和一种新型寡糖(龙胆二糖)脱氢酶(KiOdhA)。值得注意的是,发现了两种脱氢酶（TaGdhA和KiOdhA）具有利用苯氧基自由基作为电子受体的能力。有趣的是,当用作两种氧化酶（ApGoxA和PsAaoA）的电子受体时，发现苯氧基自由基在有氧环境中与分子氧竞争，这揭示了它们的多功能性。此外，基于AlphaFold生成的同源性模型，讨论了控制其不同酶功能的分子决定簇。
结论：AA3_2s的系统发育分析和生化表征为AA3_2序列和蛋白质的未来研究提供了有价值的指导。观察到酶功能与SSN聚类之间的明显相关性。这些新的AA3_2氧化还原酶的发现和生化表征为生物技术应用带来了令人兴奋的前景，并拓宽了我们对其生物学功能的理解。
结果：在这项研究中，采用序列相似性网络（SSN）和最大似然树的系统发育分析用于对AA3_2序列进行聚类。选择代表不同簇的总共27种AA3_2蛋白用于重组生产。其中,成功生产了7种新的AA3_2氧化还原酶，纯化,和特点。这些酶包括两种葡萄糖脱氢酶（TaGdhA和McGdhA），一种葡萄糖氧化酶（ApGoxA），一种芳基醇氧化酶（PsAaoA），两种芳基醇脱氢酶（AsAadhA和AsAadhB），和一种新型寡糖(龙胆二糖)脱氢酶(KiOdhA)。值得注意的是,发现了两种脱氢酶（TaGdhA和KiOdhA）具有利用苯氧基自由基作为电子受体的能力。有趣的是,当用作两种氧化酶（ApGoxA和PsAaoA）的电子受体时，发现苯氧基自由基在有氧环境中与分子氧竞争，这揭示了它们的多功能性。此外，基于AlphaFold生成的同源性模型，讨论了控制其不同酶功能的分子决定簇。
结论：AA3_2s的系统发育分析和生化表征为AA3_2序列和蛋白质的未来研究提供了有价值的指导。观察到酶功能与SSN聚类之间的明显相关性。这些新的AA3_2氧化还原酶的发现和生化表征为生物技术应用带来了令人兴奋的前景，并拓宽了我们对其生物学功能的理解。
