Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.
Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada.
Appl Environ Microbiol. 2024 Jul 24;90(7):e0101424. doi: 10.1128/aem.01014-24. Epub 2024 Jul 2.
Bacterial and fungal copper radical oxidases (CROs) from Auxiliary Activity Family 5 (AA5) are implicated in morphogenesis and pathogenesis. The unique catalytic properties of CROs also make these enzymes attractive biocatalysts for the transformation of small molecules and biopolymers. Despite a recent increase in the number of characterized AA5 members, especially from subfamily 2 (AA5_2), the catalytic diversity of the family as a whole remains underexplored. In the present study, phylogenetic analysis guided the selection of six AA5_2 members from diverse fungi for recombinant expression in (syn. ) and biochemical characterization . Five of the targets displayed predominant galactose 6-oxidase activity (EC 1.1.3.9), and one was a broad-specificity aryl alcohol oxidase (EC 1.1.3.7) with maximum activity on the platform chemical 5-hydroxymethyl furfural (EC 1.1.3.47). Sequence alignment comparing previously characterized AA5_2 members to those from this study indicated various amino acid substitutions at active site positions implicated in the modulation of specificity.IMPORTANCEEnzyme discovery and characterization underpin advances in microbial biology and the application of biocatalysts in industrial processes. On one hand, oxidative processes are central to fungal saprotrophy and pathogenesis. On the other hand, controlled oxidation of small molecules and (bio)polymers valorizes these compounds and introduces versatile functional groups for further modification. The biochemical characterization of six new copper radical oxidases further illuminates the catalytic diversity of these enzymes, which will inform future biological studies and biotechnological applications.
辅助活性家族 5（AA5）的细菌和真菌铜自由基氧化酶（CRO）与形态发生和发病机制有关。CRO 的独特催化特性也使这些酶成为小分子和生物聚合物转化的有吸引力的生物催化剂。尽管最近表征的 AA5 成员数量有所增加，尤其是亚家族 2（AA5_2），但整个家族的催化多样性仍未得到充分探索。在本研究中，系统发育分析指导从不同真菌中选择六个 AA5_2 成员进行重组表达和生化特性分析。五个目标表现出主要的半乳糖 6-氧化酶活性（EC 1.1.3.9），一个是广泛特异性的芳醇氧化酶（EC 1.1.3.7），在平台化学物质 5-羟甲基糠醛（EC 1.1.3.47）上具有最大活性。将以前表征的 AA5_2 成员与本研究中的成员进行序列比对表明，活性位点位置的各种氨基酸取代与特异性的调节有关。
重要性
酶的发现和表征是微生物生物学进展和生物催化剂在工业过程中应用的基础。一方面，氧化过程是真菌腐生和发病机制的核心。另一方面，小分子和（生物）聚合物的可控氧化使这些化合物具有更高的价值，并引入了多功能基团，以进一步进行修饰。对六种新的铜自由基氧化酶的生化特性进行了表征，进一步阐明了这些酶的催化多样性，这将为未来的生物学研究和生物技术应用提供信息。
