Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea.
Biosciences Division, Structural Biology Center, Argonne National Laboratory, Argonne, Illinois, USA.
Acta Crystallogr D Struct Biol. 2023 Dec 1;79(Pt 12):1094-1108. doi: 10.1107/S2059798323009609.
Cyanase plays a vital role in the detoxification of cyanate and supplies a continuous nitrogen source for soil microbes by converting cyanate to ammonia and carbon dioxide in a bicarbonate-dependent reaction. The structures of cyanase complexed with dianion inhibitors, in conjunction with biochemical studies, suggest putative binding sites for substrates. However, the substrate-recognition and reaction mechanisms of cyanase remain unclear. Here, crystal structures of cyanase from Escherichia coli were determined in the native form and in complexes with cyanate, bicarbonate and intermediates at 1.5-1.9 Å resolution using synchrotron X-rays and an X-ray free-electron laser. Cyanate and bicarbonate interact with the highly conserved Arg96, Ser122 and Ala123 in the active site. In the presence of a mixture of cyanate and bicarbonate, three different electron densities for intermediates were observed in the cyanase structures. Moreover, the observed electron density could explain the dynamics of the substrate or product. In addition to conformational changes in the substrate-binding pocket, dynamic movement of Leu151 was observed, which functions as a gate for the passage of substrates or products. These findings provide a structural mechanism for the substrate-binding and reaction process of cyanase.
氰酸酶在氰酸盐解毒中起着至关重要的作用，通过在碳酸氢盐依赖的反应中将氰酸盐转化为氨和二氧化碳，为土壤微生物提供持续的氮源。结合生化研究，氰酸酶与二价阴离子抑制剂形成的复合物的结构提示了可能的底物结合位点。然而，氰酸酶的底物识别和反应机制仍不清楚。在这里，使用同步辐射 X 射线和自由电子激光，以 1.5-1.9Å 的分辨率测定了大肠杆菌氰酸酶的天然形式和与氰酸盐、碳酸氢盐和中间体复合物的晶体结构。氰酸盐和碳酸氢盐与高度保守的 Arg96、Ser122 和 Ala123 在活性位点相互作用。在氰酸盐和碳酸氢盐的混合物存在下，在氰酸酶结构中观察到三种不同的中间体的电子密度。此外，观察到的电子密度可以解释底物或产物的动力学。除了底物结合口袋的构象变化外，还观察到 Leu151 的动态运动，它作为底物或产物通过的门。这些发现为氰酸酶的底物结合和反应过程提供了结构机制。
