Polyamines are bioactive amines that play a variety of roles, such as promoting cell proliferation and protein synthesis, and the intestinal lumen contains up to several mM polyamines derived from the gut microbiota. In the present study, we conducted genetic and biochemical analyses of the polyamine biosynthetic enzyme N-carbamoylputrescine amidohydrolase (NCPAH) that converts N-carbamoylputrescine to putrescine, a precursor of spermidine in Bacteroides thetaiotaomicron, which is one of the most dominant species in the human gut microbiota. First, ncpah gene deletion and complemented strains were generated, and the intracellular polyamines of these strains cultured in a polyamine-free minimal medium were analyzed using high-performance liquid chromatography. The results showed that spermidine detected in the parental and complemented strains was depleted in the gene deletion strain. Next, purified NCPAH-(His)6 was analyzed for enzymatic activity and found to be capable of converting N-carbamoylputrescine to putrescine, with a Michaelis constant (Km) and turnover number (kcat) of 730 µM and 0.8 s−1, respectively. Furthermore, the NCPAH activity was strongly (>80%) inhibited by agmatine and spermidine, and moderately (≈50%) inhibited by putrescine. This feedback inhibition regulates the reaction catalyzed by NCPAH and may play a role in intracellular polyamine homeostasis in B. thetaiotaomicron.

中文翻译：

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拟杆菌的 N-氨基甲酰腐胺酰胺水解酶，一种人类肠道微生物群的优势种

多胺是具有多种作用的生物活性胺，例如促进细胞增殖和蛋白质合成，肠腔含有多达数 mM 来自肠道微生物群的多胺。在本研究中，我们对多胺生物合成酶 N-氨基甲酰腐胺酰胺水解酶 (NCPAH) 进行了遗传和生化分析，该酶将 N-氨基甲酰腐胺转化为腐胺，后者是多形拟杆菌中亚精胺的前体，是人类最主要的物种之一肠道菌群。首先，生成ncpah基因缺失和互补菌株，并使用高效液相色谱分析在无多胺基本培养基中培养的这些菌株的细胞内多胺。结果表明，在亲本和互补菌株中检测到的亚精胺在基因缺失菌株中被耗尽。接下来，分析纯化的 NCPAH-(His)6 的酶活性，发现它能够将 N-氨基甲酰腐胺转化为腐胺，其米氏常数 (Km) 和周转数 (kcat) 分别为 730 µM 和 0.8 s−1 . 此外，NCPAH 活性被胍丁胺和亚精胺强烈抑制 (>80%)，并被腐胺中度抑制 (≈50%)。这种反馈抑制调节 NCPAH 催化的反应，并可能在 B.thetaiotaomicron 的细胞内多胺稳态中发挥作用。米氏常数 (Km) 和周转数 (kcat) 分别为 730 µM 和 0.8 s−1。此外，NCPAH 活性被胍丁胺和亚精胺强烈抑制 (>80%)，并被腐胺中度抑制 (≈50%)。这种反馈抑制调节 NCPAH 催化的反应，并可能在 B.thetaiotaomicron 的细胞内多胺稳态中发挥作用。米氏常数 (Km) 和周转数 (kcat) 分别为 730 µM 和 0.8 s−1。此外，NCPAH 活性被胍丁胺和亚精胺强烈抑制 (>80%)，并被腐胺中度抑制 (≈50%)。这种反馈抑制调节 NCPAH 催化的反应，并可能在 B.thetaiotaomicron 的细胞内多胺稳态中发挥作用。