Microbiology and Immunology, The University of British Columbia, Vancouver, BC, Canada.
Microbiology and Immunology, The University of British Columbia, Vancouver, BC, Canada.
Methods Enzymol. 2024;703:65-85. doi: 10.1016/bs.mie.2024.05.016. Epub 2024 Jun 18.
Oxygenases catalyze crucial reactions throughout all domains of life, cleaving molecular oxygen (O) and inserting one or two of its atoms into organic substrates. Many oxygenases, including those in the cytochrome P450 (P450) and Rieske oxygenase enzyme families, function as multicomponent systems, which require one or more redox partners to transfer electrons to the catalytic center. As the identity of the reductase can change the reactivity of the oxygenase, characterization of the latter with its cognate redox partners is critical. However, the isolation of the native redox partner or partners is often challenging. Here, we report the preparation and characterization of PbdB, the native reductase partner of PbdA, a bacterial P450 enzyme that catalyzes the O-demethylation of para-methoxylated benzoates. Through production in a rhodoccocal host, codon optimization, and anaerobic purification, this procedure overcomes conventional challenges in redox partner production and allows for robust oxygenase characterization with its native redox partner. Key lessons learned here, including the value of production in a related host and rare codon effects are applicable to a broad range of Fe-dependent oxygenases and their components.
加氧酶在所有生命领域中催化关键反应，裂解分子氧（O）并将其一个或两个原子插入有机底物中。许多加氧酶，包括细胞色素 P450（P450）和 Rieske 加氧酶酶家族中的那些，作为多组分系统起作用，这些系统需要一个或多个氧化还原伴侣将电子转移到催化中心。由于还原酶的身份可以改变加氧酶的反应性，因此对其具有同源氧化还原伴侣的后者进行特征描述至关重要。然而，天然氧化还原伴侣的分离通常具有挑战性。在这里，我们报告了 PbdB 的制备和表征，PbdB 是 PbdA 的天然还原酶伴侣，PbdA 是一种细菌 P450 酶，可催化对甲氧基苯甲酸的 O-去甲基化。通过在红色球菌宿主中的生产、密码子优化和厌氧纯化，该程序克服了氧化还原伴侣生产中的常规挑战，并允许与天然氧化还原伴侣进行强大的加氧酶特征描述。在这里学到的关键经验教训，包括在相关宿主中生产的价值和稀有密码子效应，适用于广泛的 Fe 依赖性加氧酶及其成分。
