Guanine deaminases (GDs) are essential enzymes that regulate the overall nucleobase pool. Since the deamination of guanine to xanthine results in the production of a mutagenic base, these enzymes have evolved to be very specific in nature. Surprisingly, they accept structurally distinct triazine ammeline, an intermediate in the melamine pathway, as one of the moonlighting substrates. Here, by employing NE0047 (a GD from Nitrosomonas europaea), we delineate the nuance in the catalytic mechanism that allows these two distinct substrates to be catalyzed. A combination of enzyme kinetics, X-ray crystallographic, and calorimetric studies reveal that GDs operate via a dual proton shuttle mechanism with two glutamates, E79 and E143, crucial for deamination. Additionally, N66 appears to be central for substrate anchoring and participates in catalysis. The study highlights the importance of closure of the catalytic loop and of maintenance of the hydrophobic core by capping residues like F141 and F48 for the creation of an apt environment for activation of the zinc-assisted catalysis. This study also analyzes evolutionarily distinct GDs and asserts that GDs incorporate subtle variations in the active site architectures while keeping the most critical active site determinants conserved.

鸟嘌呤脱氨酶 (GDs) 是调节整个核碱基池的必需酶。由于鸟嘌呤脱氨基生成黄嘌呤会导致产生诱变碱基，因此这些酶在性质上已进化为非常专一。令人惊讶的是，他们接受结构上不同的三嗪三聚氰胺（三聚氰胺途径的中间体）作为兼职基质之一。在这里，通过使用 NE0047（来自欧洲亚硝化单胞菌的 GD)，我们描述了催化机制中的细微差别，它允许这两种不同的底物被催化。酶动力学、X 射线晶体学和量热研究的结合表明，GDs 通过双质子穿梭机制与两种谷氨酸 E79 和 E143 共同作用，这对于脱氨基作用至关重要。此外，N66 似乎是底物锚定的中心并参与催化。该研究强调了封闭催化环和通过封端 F141 和 F48 等残基来维持疏水核心的重要性，以便为锌辅助催化的活化创造适宜的环境。本研究还分析了进化上不同的 GDs，并断言 GDs 在活动位点架构中包含细微的变化，同时保持最关键的活动位点决定因素的保守。