The sulfolipid sulfoquinovosyl diacylglycerol (SQDG), produced by plants, algae, and cyanobacteria, constitutes a major sulfur reserve in the biosphere. Microbial breakdown of SQDG is critical for the biological utilization of its sulfur. This commences through release of the parent sugar, sulfoquinovose (SQ), catalyzed by sulfoquinovosidases (SQases). These vanguard enzymes are encoded in gene clusters that code for diverse SQ catabolic pathways. To identify, visualize and isolate glycoside hydrolase CAZY‐family 31 (GH31) SQases in complex biological environments, we introduce SQ cyclophellitol‐aziridine activity‐based probes (ABPs). These ABPs label the active site nucleophile of this enzyme family, consistent with specific recognition of the SQ cyclophellitol‐aziridine in the active site, as evidenced in the 3D structure of Bacillus megaterium SQase. A fluorescent Cy5‐probe enables visualization of SQases in crude cell lysates from bacteria harbouring different SQ breakdown pathways, whilst a biotin‐probe enables SQase capture and identification by proteomics. The Cy5‐probe facilitates monitoring of active SQase levels during different stages of bacterial growth which show great contrast to more traditional mRNA analysis obtained by RT‐qPCR. Given the importance of SQases in global sulfur cycling and in human microbiota, these SQase ABPs provide a new tool with which to study SQase occurrence, activity and stability.

中文翻译：

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使用基于活性的探针检测细胞裂解物中的磺基喹诺糖苷酶活性

由植物、藻类和蓝藻产生的硫脂磺基喹诺酰二酰基甘油 (SQDG) 构成了生物圈中的主要硫储备。 SQDG 的微生物分解对其硫的生物利用至关重要。这是通过磺基奎诺糖苷酶 (SQase) 催化的母体糖磺基奎诺糖 (SQ) 的释放开始的。这些先锋酶被编码在编码不同 SQ 分解代谢途径的基因簇中。为了在复杂的生物环境中识别、可视化和分离糖苷水解酶 CAZY 家族 31 (GH31) SQase，我们引入了 SQ 环己醇-氮丙啶活性探针 (ABP)。这些 ABP 标记了该酶家族的活性位点亲核体，与活性位点中 SQ 环苯乙醇-氮丙啶的特异性识别一致，如巨大芽孢杆菌 SQase 的 3D 结构所示。荧光 Cy5 探针能够对来自具有不同 SQ 分解途径的细菌的粗细胞裂解物中的 SQase 进行可视化，而生物素探针则能够通过蛋白质组学捕获和识别 SQase。 Cy5 探针有助于监测细菌生长不同阶段的活性 SQase 水平，这与通过 RT-qPCR 获得的更传统的 mRNA 分析形成鲜明对比。鉴于 SQase 在全球硫循环和人类微生物群中的重要性，这些 SQase ABP 提供了一种新工具来研究 SQase 的发生、活性和稳定性。