Department of Biological Science and Engineering, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 100083, Beijing, China.
Department of Biological Science and Engineering, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 100083, Beijing, China.
Chemosphere. 2019 Dec;236:124281. doi: 10.1016/j.chemosphere.2019.07.012. Epub 2019 Jul 2.
Microcystinase (MlrA) catalyzes the first and most important biodegradation step of hepatotoxic microcystin-LR (MC-LR) produced and released from cyanobacterial cells, and the underlying catalytic mechanism is not completely understood yet. MlrA was postulated previously to be a metalloprotease with an active site of HAIHNE, a variant of the common metal-binding motif of HEXXH. Through comparison with representative modes in HEXXH-containing metalloproteases, molecular dynamics simulation, homology modeling, and docking, the active sites of MlrA involved in the MC-LR biodegradation by Sphingomonas sp. USTB-05 were predicted. Site-directed mutants of MlrA were constructed for verification then. The results show that MlrA is likely not a metalloprotease, but a glutamate protease belonging to type II CAAX prenyl endopeptidases. Combined with the biodegradation of MC-LR by MlrA and its mutants, a complete enzymatic mechanism for MC-LR biodegradation by MlrA is proposed: Glu and His activate a water molecule facilitating a nucleophilic attack on the Adda-Arg peptide bond of MC-LR; Trp and Trp contact the carboxylate side chain of Gluand, by raising its pKa potentially, accelerate the reaction rates; His and Asn (located in the previous postulated active center of HAIHNE) function as an oxyanion hole to stabilize the transition states. This study reveals the enzymatic mechanism of MlrA for catalyzing MC-LR in both the representative modes and the experiments of site-directed mutagenesis.
微囊藻酶（MlrA）催化从蓝藻细胞中产生和释放的肝毒性微囊藻-LR（MC-LR）的第一个也是最重要的生物降解步骤，但其潜在的催化机制尚不完全清楚。先前推测 MlrA 是一种金属蛋白酶，其活性位点为 HAIHNE，这是 HEXXH 常见金属结合基序的变体。通过与含 HEXXH 的金属蛋白酶的代表性模式进行比较、分子动力学模拟、同源建模和对接，预测了 Sphingomonas sp. USTB-05 中参与 MC-LR 生物降解的 MlrA 的活性位点。然后构建了 MlrA 的定点突变体进行验证。结果表明，MlrA 可能不是金属蛋白酶，而是属于 II 型 CAAX prenyl endopeptidases 的谷氨酸蛋白酶。结合 MlrA 及其突变体对 MC-LR 的生物降解，提出了 MlrA 对 MC-LR 生物降解的完整酶促机制：Glu 和 His 激活水分子，有利于对 MC-LR 的 Adda-Arg 肽键进行亲核攻击；Trp 和 Trp 与 Glu 的羧酸盐侧链接触，通过提高其 pKa 可能加速反应速率；His 和 Asn（位于先前推测的 HAIHNE 活性中心）作为一个氧阴离子穴，稳定过渡态。本研究揭示了 MlrA 以代表性模式和定点突变实验催化 MC-LR 的酶促机制。
