School of Environmental Studies, Jadavpur University, Kolkata 700 032, India.
Department of Biotechnology, Indian Institute of Technology (Roorkee), Roorkee 247 667, India.
Int J Biol Macromol. 2019 Mar 1;124:635-650. doi: 10.1016/j.ijbiomac.2018.11.206. Epub 2018 Nov 23.
A protease of the primary pathogen (Pseudoalteromonas agarivorans NW4327) of the disease affecting the Great Barrier Reef sponge Rhopaloeides odorabile was purified. Zymography demonstrated calcium-dependent collagenase and gelatinase activity of the purified protein. This metalloprotease was identified by matrix assisted laser desorption ionization time-of-flight mass spectrophotometry as a 52,509 Da U32 collagenase. Predicted tertiary structure of U32 collagenase (by Phyre2 fold recognition server) demonstrated 13% identity with known hydrolases establishing novelty of the enzyme. Molecular docking conceived two interacting loops of the collagenase that bound with collagen triple helices and two calcium ions remained centered between the loops. According to ConSurf multiple sequence alignment, the residues of loop1 of the collagenase were mostly conserved while variations among residues of loop2 were comparatively higher than loop1. Asp262, Glu263 of loop1 and Thr363, Lys364, Gln365 of loop2 participated in the interaction with Ca and collagen. Root mean square deviation and root mean square fluctuation values signified higher stability of the collagen-Ca-collagenase complex and greater structural stability of the residues of the loops in the complex compared to apocollagenase. Observed properties of NW4327 U32 collagenase and its interaction with collagen were different from similar enzymes of thermophilic bacteria and terrestrial pathogens.
从影响大堡礁海绵（Rhopaloeides odorabile）疾病的主要病原体（假交替单胞菌 NW4327）中纯化出一种蛋白酶。酶谱分析表明，纯化蛋白具有钙依赖性胶原蛋白酶和明胶酶活性。基质辅助激光解吸电离飞行时间质谱分析将这种金属蛋白酶鉴定为 52,509 Da 的 U32 胶原蛋白酶。通过 Phyre2 折叠识别服务器预测的 U32 胶原蛋白酶的三级结构与已知的水解酶具有 13%的同一性，确立了该酶的新颖性。分子对接设想胶原蛋白酶的两个相互作用环与胶原蛋白三螺旋结合，两个钙离子仍位于环之间的中心。根据 ConSurf 多序列比对，胶原蛋白酶环 1 的残基大多保守，而环 2 的残基之间的变异比环 1 高。环 1 的 Asp262、Glu263 和环 2 的 Thr363、Lys364、Gln365 参与了与 Ca 和胶原蛋白的相互作用。均方根偏差和均方根波动值表明胶原-Ca-胶原蛋白酶复合物具有更高的稳定性，并且与原胶原蛋白酶相比，复合物中环的残基具有更大的结构稳定性。NW4327 U32 胶原蛋白酶及其与胶原蛋白的相互作用性质与嗜热菌和陆地病原体的类似酶不同。
