Gao Yu, Zheng Lanyan, Li Jian-Jun, Du Yuguang
Department of Microbiology and Parasitology, China Medical University, No.77 Puhe Road, Shenyang 110122, Liaoning Province, China.
Department of Microbiology and Parasitology, China Medical University, No.77 Puhe Road, Shenyang 110122, Liaoning Province, China.
Arch Biochem Biophys. 2016 Dec 15;612:9-16. doi: 10.1016/j.abb.2016.10.004. Epub 2016 Oct 5.
Two structural Ca (proximal and distal) is known to be important for ligninolytic peroxidases. However, few studies toward impact of residues involved in two Ca on properties of ligninolytic peroxidases have been done, especially the proximal one. In this study, mutants of nine residues involved in liganding two Ca of Pleurotus eryngii versatile peroxidase (VP) were investigated. Most mutants almost completely lost activities, except the mutants of proximal Ca - S170A and V192T. In comparison with WT (wild type), optimal pH values of S170A, S170D, and V192T shifted from pH 3.0 to pH 3.5. The order of thermal and pH stabilities of WT, V192T, S170A, and S170D is similar to that of their specific activities: WT > V192T > S170A > S170D. The CD (circular dichroism) results of WT and several mutants indicated that mutations had some effects on secondary structures. For the first time, it was observed that the thermostability of ligninolytic peroxidases is related with proximal Ca too, and the mutant containing distal Ca only was obtained. Our results clearly demonstrated that enzymatic activities, pH and thermal stabilities, Cacontent, and secondary structures of VP have close relationship with the residues involved in two structural Ca.
已知两个结构钙（近端和远端）对木质素分解过氧化物酶很重要。然而，针对参与两个钙的残基对木质素分解过氧化物酶性质影响的研究很少，尤其是近端的那个。在本研究中，对杏鲍菇多功能过氧化物酶（VP）中参与结合两个钙的九个残基的突变体进行了研究。除了近端钙的突变体S170A和V192T外，大多数突变体几乎完全丧失了活性。与野生型（WT）相比，S170A、S170D和V192T的最佳pH值从pH 3.0变为pH 3.5。WT、V192T、S170A和S170D的热稳定性和pH稳定性顺序与其比活性顺序相似：WT > V192T > S170A > S170D。WT和几个突变体的圆二色性（CD）结果表明，突变对二级结构有一些影响。首次观察到木质素分解过氧化物酶的热稳定性也与近端钙有关，并且获得了仅含有远端钙的突变体。我们的结果清楚地表明，VP的酶活性、pH和热稳定性、钙含量以及二级结构与参与两个结构钙的残基密切相关。
