The 1.5 Å resolution crystal structure of the carbamate kinase-like carbamoyl phosphate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus, bound to ADP, confirms that this thermostable enzyme is a carbamate kinase, and provides insight into substrate binding and stability in carbamate kinases
The 1.5 Å resolution crystal structure of the carbamate kinase-like carbamoyl phosphate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus, bound to ADP, confirms that this thermostable enzyme is a carbamate kinase, and provides insight into substrate binding and stability in carbamate kinases
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DOI：
10.1006/jmbi.2000.3779
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发表时间：
2000-06-02
影响因子：
5.6
通讯作者：
Rubio, V
中科院分区：
文献类型：
--
作者：
Ramón-Maiques, S;Marina, A;Rubio, V
氨基甲酰磷酸 (CP) 是精氨酸和嘧啶碱基的重要前体，由 CP 合成酶 (CPS) 分三步合成。最后一步，氨基甲酸酯的磷酸化，也由氨基甲酸酯激酶 (CK) 催化，CK 是微生物用来从 ADP 和 CP 产生 Am 的酶。尽管最近确定的 CPS 和 CK 结构没有显示出明显的相互相似性，但在超嗜热古细菌中报道的类似 CK 的 CPS 被认为是 CP 生物合成进化中缺失的环节。这种来自激烈火球菌的酶的 1.5 埃分辨率结构显示出亚基拓扑结构和同二聚体分子组织，具有被 α 螺旋包围的 16 链开放 β 折叠核心，与 CK 中的类似。然而，火球菌酶表现出许多溶剂可接近的离子对、广泛的、强疏水性的亚基间表面，并呈现结合的 ADP 分子，该分子在 22°C 时不会从酶解离。 ADP 核苷酸被隔离在大空腔底部的核心层 C 边缘上形成的脊中，嘌呤环封闭在腺嘌呤特异性的口袋中。总体而言，该酶结构不适合催化 CPS 的特征性三步反应，并支持这样的观点，即 CK 样 CPS 实际上是一种高度耐热且非常缓慢（37 摄氏度）的 CK，在 P. Furiosus 的极端环境中，可能具有制造而不是使用 CP 的新功能。酶的热稳定性可能是由于疏水亚基间接触的延伸和大量暴露的离子对造成的，其中一些离子对在每个酶亚基的几个二级结构元件上形成离子对网络。该结构提供了有关 CK 中底物结合和催化作用的第一个信息，并表明：37 摄氏度下的缓慢速率可能是产物解离缓慢的结果。(C) 2000 学术出版社。
Carbamoyl phosphate (CP), an essential precursor of arginine and the pyrimidine bases, is synthesized by CP synthetase (CPS) in three steps. The last step, the phosphorylation of carbamate, is also catalyzed by carbamate kinase (CK), an enzyme used by microorganisms to produce Am from ADP and CP. Although the recently determined structures of CPS and CK show no obvious mutual similarities, a CK-like CPS reported in hyperthermophilic archaea was postulated to be a missing Link in the evolution of CP biosynthesis. The 1.5 Angstrom resolution structure of this enzyme from Pyrococcus furiosus shows both a subunit topology and a homodimeric molecular organization, with a 16-stranded open beta-sheet core surrounded by alpha-helices, similar to those in CK. However, the pyrococcal enzyme exhibits many solvent-accessible ion-pairs, an extensive, strongly hydrophobic, intersubunit surface, and presents a bound,ADP molecule, which does not dissociate at 22 degrees C from the enzyme. The ADP nucleotide is sequestered in a ridge formed over the C-edge of the core sheet, at the bottom of a large cavity, with the purine ring enclosed in a pocket specific for adenine. Overall, the enzyme structure is ill-suited for catalyzing the characteristic three-step reaction of CPS and supports the view that the CK-Like CPS is in fact a highly thermostable and very slow (at 37 degrees C) CK that, in the extreme environment of P. furiosus, may have the new function of making, rather than using, CP. The thermostability of the enzyme may result from the extension of the hydrophobic intersubunit contacts and from the large number of exposed ion-pairs, some of which form ion-pair net-works across several secondary structure elements in each enzyme subunit. The structure provides the first information on substrate binding and catalysis in CKs, and suggests thar: the slow rate at 37 degrees C is possibly a consequence of slow product dissociation.(C) 2000 Academic Press.