Free guanidine is increasingly recognized as a relevant molecule in biological systems. Recently, it was reported that urea carboxylase acts preferentially on guanidine, and consequently, it was considered to participate directly in guanidine biodegradation. Urea carboxylase combines with allophanate hydrolase to comprise the activity of urea amidolyase, an enzyme predominantly found in bacteria and fungi that catalyzes the carboxylation and subsequent hydrolysis of urea to ammonia and carbon dioxide. Here, we demonstrate that urea carboxylase and allophanate hydrolase from Pseudomonas syringae are insufficient to catalyze the decomposition of guanidine. Rather, guanidine is decomposed to ammonia through the combined activities of urea carboxylase, allophanate hydrolase, and two additional proteins of the DUF1989 protein family, expansively annotated as urea carboxylase-associated family proteins. These proteins comprise the subunits of a heterodimeric carboxyguanidine deiminase (CgdAB), which hydrolyzes carboxyguanidine to N-carboxyurea (allophanate). The genes encoding CgdAB colocalize with genes encoding urea carboxylase and allophanate hydrolase. However, 25% of urea carboxylase genes, including all fungal urea amidolyases, do not colocalize with cgdAB. This subset of urea carboxylases correlates with a notable Asp to Asn mutation in the carboxyltransferase active site. Consistent with this observation, we demonstrate that fungal urea amidolyase retains a strong substrate preference for urea. The combined activities of urea carboxylase, carboxyguanidine deiminase and allophanate hydrolase represent a newly recognized pathway for the biodegradation of guanidine. These findings reinforce the relevance of guanidine as a biological metabolite and reveal a broadly distributed group of enzymes that act on guanidine in bacteria.

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解决难题：广泛用于细菌中尿素代谢注释的蛋白质是介导胍氮同化的羧基胍脱亚胺酶。

游离胍越来越多地被认为是生物系统中的相关分子。最近，有报道称尿素羧化酶优先作用于胍，因此被认为直接参与了胍的生物降解。尿素羧化酶与脲基甲酸酯水解酶结合以包含尿素酰胺分解酶的活性，该酶主要存在于细菌和真菌中，催化尿素羧化和随后水解成氨和二氧化碳。在这里，我们证明了来自丁香假单胞菌的尿素羧化酶和脲基甲酸酯水解酶不足以催化胍的分解。相反，胍通过尿素羧化酶、脲基甲酸酯水解酶和 DUF1989 蛋白家族的两种其他蛋白质（广泛注释为尿素羧化酶相关家族蛋白）的联合活动分解为氨。这些蛋白质包含异二聚体羧基胍脱亚胺酶 (CgdAB) 的亚基，可将羧基胍水解为N-羧基脲（脲基甲酸酯）。编码 CgdAB 的基因与编码尿素羧化酶和脲基甲酸水解酶的基因共定位。然而，25% 的尿素羧化酶基因，包括所有真菌尿素酰胺酶，不与cgdAB共定位. 该尿素羧化酶亚群与羧基转移酶活性位点中显着的 Asp 至 Asn 突变相关。与这一观察结果一致，我们证明真菌尿素酰胺酶保留对尿素的强烈底物偏好。尿素羧化酶、羧基胍脱亚胺酶和脲基甲酸酯水解酶的联合活性代表了一种新发现的胍生物降解途径。这些发现加强了胍作为生物代谢物的相关性，并揭示了一组广泛分布的酶，这些酶作用于细菌中的胍。