The high-resolution crystal structure of lobster hemocyanin shows its enzymatic capability as a phenoloxidase.
The high-resolution crystal structure of lobster hemocyanin shows its enzymatic capability as a phenoloxidase.
复制标题
DOI：
10.1016/j.abb.2020.108370
复制
发表时间：
2020-05
影响因子：
3.9
通讯作者：
T. Masuda;S. Baba;K. Matsuo;S. Ito;B. Mikami
中科院分区：
文献类型：
--
作者：
T. Masuda;S. Baba;K. Matsuo;S. Ito;B. Mikami
血蓝蛋白 (Hc) 和酚氧化酶 (PO) 是 3 型铜蛋白家族的成员。尽管节肢动物 Hc 和 PO 表现出相似的含铜活性位点三维结构，但 Hc 作为氧转运蛋白发挥作用，表现出最小的酚氧化酶活性或没有酚氧化酶活性。在这里，我们以 1.58 Å 的分辨率展示了日本龙虾 (Panulirus japonicus) 的 Hc 氧型晶体结构 (PjHc)。发现 PjHc 的双铜活性位点的结构与 PO 的结构几乎相同。尽管在 PjHc 中与 PO 几乎相同的位置观察到对酶活性至关重要的保守氨基酸和水分子，但 PjHc 在我们的实验条件下没有显示出酶活性。 PjHc 和节肢动物 PO 之间的一个显着差异是 PjHc 双核铜位点附近存在“阻断剂残留物”。该阻断剂残基包含与 CuA 配位组氨酸的咪唑环紧密堆叠的苯丙氨酸残基，并阻碍底物进入活性位点。我们的结果表明，阻断剂残基也是 3 型铜蛋白催化活性的决定因素。
Hemocyanin (Hc) and phenoloxidase (PO) are members of the type 3 copper protein family. Although arthropod Hc and PO exhibit similar three-dimensional structures of the copper-containing active site, Hc functions as an oxygen transport protein, showing minimal or no phenoloxidase activity. Here, we present the crystal structure of the oxy form of Hc fromPanulirus japonicus(PjHc) at 1.58 Å resolution. The structure of the di-copper active site of PjHc was found to be almost identical to that of PO. Although conserved amino acids and the water molecule crucial for the enzymatic activity were observed in PjHc at almost the same positions as those in PO, PjHc showed no enzymatic activity under our experimental conditions. One striking difference between PjHc and arthropod PO was the presence of a “blocker residue” near the binuclear copper site of PjHc. This blocker residue comprised a phenylalanine residue tightly stacked with an imidazole ring of a CuA coordinated histidine and hindered substrates from accessing the active site. Our results suggest that the blocker residue is also a determining factor of the catalytic activity of type 3 copper proteins.