Vats Anshika, Anas Shukurah, Chakraborty Ankush, Liu Jian, Ryu Jimyung, Mann Sara M, Andorfer Mary C
Department of Chemistry, Michigan State University, East Lansing, MI, USA.
Bio Protoc. 2025 Jun 20;15(12):e5357. doi: 10.21769/BioProtoc.5357.
X-succinate synthase enzymes (XSSs) are a class of glycyl radical enzymes (GREs) that play a pivotal role in microbial anaerobic hydrocarbon degradation. They catalyze the addition of hydrocarbons to fumarate using a protein-based glycyl radical, which must first be installed by a radical -adenosylmethionine (rSAM) activating enzyme (AE). Once activated, XSS enzymes can undergo multiple catalytic cycles, forming C(sp)-C(sp) bonds with high stereoselectivity-a feature that highlights their potential as asymmetric biocatalysts. Due to the insolubility of XSS-AEs when heterologously expressed in , studies have relied on in vivo radical installation protocols. Although these methods have illuminated fundamental details of XSS mechanisms, the inability to install a glycyl radical in vitro has limited biochemical studies and biotechnological advances using these enzymes. Here, we describe an in vitro protocol for reconstituting the activity of benzylsuccinate synthase (BSS), an XSS that catalyzes the addition of toluene to fumarate to form -benzylsuccinate. To enable in vitro glycyl radical installation, we identified a soluble homolog via genome mining: 4-isopropylbenzylsuccinate synthase activating enzyme (IbsAE). IbsAE was expressed in and anaerobically purified in moderate yields (6-8 mg of protein per liter of culture); herein, we outline the expression and anaerobic purification of both IbsAE and BSS proteins. We describe a reproducible method for in vitro glycyl radical installation using these recombinant proteins and provide guidance on quantifying radical formation. Our optimized protocol consistently achieves 30%-50% radical installation, comparable to other in vitro GRE activations. Lastly, we demonstrate the application of this protocol for in vitro hydroalkylation reactions, achieving high assay yields (89%-97%). This protocol enables biochemical experiments that were previously challenging using cell extracts and accelerated advancements in XSS engineering and use in biocatalysis. Key features • Builds upon the method described by Andorfer et al. [1] to thoroughly describe in vitro activation and hydroalkylation using glycyl radical enzymes. • Useful for studying substrate scope and determining the stereoselectivity of XSS-catalyzed reactions with non-native substrates. • Can serve as a template for the reconstitution of activity for other XSS enzymes. • Describes protein production through hydroalkylation steps, which take approximately 6-7 days to complete, given that expressions and purifications are performed in parallel.
X-琥珀酸合酶（XSSs）是一类甘氨酰自由基酶（GREs），在微生物厌氧烃降解中起关键作用。它们利用基于蛋白质的甘氨酰自由基催化烃类与富马酸的加成反应，而该自由基必须首先由自由基 - 腺苷甲硫氨酸（rSAM）激活酶（AE）安装。一旦被激活，XSS酶可以进行多个催化循环，以高立体选择性形成C(sp)-C(sp)键——这一特性突出了它们作为不对称生物催化剂的潜力。由于XSS-AE在异源表达时不溶性，研究依赖于体内自由基安装方案。尽管这些方法揭示了XSS机制的基本细节，但无法在体外安装甘氨酰自由基限制了使用这些酶的生化研究和生物技术进展。在这里，我们描述了一种体外方案，用于重建苄基琥珀酸合酶（BSS）的活性，BSS是一种XSS，催化甲苯与富马酸加成形成 - 苄基琥珀酸。为了实现体外甘氨酰自由基的安装，我们通过基因组挖掘鉴定了一种可溶性同源物：4-异丙基苄基琥珀酸合酶激活酶（IbsAE）。IbsAE在 中表达，并以中等产量（每升培养物6 - 8毫克蛋白质）进行厌氧纯化；在此，我们概述了IbsAE和BSS蛋白的表达及厌氧纯化过程。我们描述了一种使用这些重组蛋白进行体外甘氨酰自由基安装的可重复方法，并提供了量化自由基形成的指导。我们优化的方案始终能实现30% - 50%的自由基安装率，与其他体外GRE激活方法相当。最后，我们展示了该方案在体外氢烷基化反应中的应用，获得了高检测产率（89% - 97%）。该方案实现了以前使用细胞提取物具有挑战性的生化实验，并加速了XSS工程及在生物催化中应用的进展。关键特性 • 基于Andorfer等人[1]描述的方法，全面描述了使用甘氨酰自由基酶的体外激活和氢烷基化过程。 • 有助于研究底物范围并确定XSS催化非天然底物反应的立体选择性。 • 可作为重建其他XSS酶活性的模板。 • 描述了从表达和纯化并行进行到氢烷基化步骤的蛋白质生产过程，整个过程大约需要6 - 7天完成。
