Xu Yang, Lv Shujing, Li Xiang, Zhai Chuanjia, Shi Yulian, Li Xuejiao, Feng Zhiyang, Luo Gan, Wang Ying, Gao Xiaoyan
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 102488, China.
J Pharm Anal. 2025 Jan;15(1):101108. doi: 10.1016/j.jpha.2024.101108. Epub 2024 Sep 21.
Sennoside A (SA), a typical prodrug, exerts its laxative effect only after its transformation into rheinanthrone catalyzed by gut microbial hydrolases and reductases. Hydrolases have been identified, but reductases remain unknown. By linking a photoreactive group to the SA scaffold, we synthesized a photoaffinity probe to covalently label SA reductases and identified SA reductases using activity-based protein profiling (ABPP). From lysates of an active strain, (. ), 397 proteins were enriched and subsequently identified using mass spectrometry (MS). Among these proteins, chromate reductase/nicotinamide adenine dinucleotide (NADH) phosphate (NADPH)-dependent flavin mononucleotide (FMN) reductase/oxygen-insensitive NADPH nitroreductase (nfrA) was identified as a potent SA reductase through further bioinformatic analysis and The Universal Protein Resource (UniProt) database screening. We also determined that recombinant nfrA could reduce SA. Our study contributes to further illuminating mechanisms of SA transformation to rheinanthrone and simultaneously offers an effective method to identify gut bacterial reductases.
番泻苷A（SA）是一种典型的前药，只有在肠道微生物水解酶和还原酶催化下转化为大黄蒽酮后才发挥其通便作用。水解酶已被鉴定出来，但还原酶仍然未知。通过将光反应基团连接到SA支架上，我们合成了一种光亲和探针，用于共价标记SA还原酶，并使用基于活性的蛋白质谱分析（ABPP）鉴定SA还原酶。从一个活性菌株的裂解物中富集了397种蛋白质，随后使用质谱（MS）进行鉴定。在这些蛋白质中，通过进一步的生物信息学分析和通用蛋白质资源（UniProt）数据库筛选，铬还原酶/烟酰胺腺嘌呤二核苷酸（NADH）磷酸（NADPH）依赖性黄素单核苷酸（FMN）还原酶/氧不敏感NADPH硝基还原酶（nfrA）被鉴定为一种有效的SA还原酶。我们还确定重组nfrA可以还原SA。我们的研究有助于进一步阐明SA转化为大黄蒽酮的机制，同时提供了一种鉴定肠道细菌还原酶的有效方法。
