Laccases (benzenediol: oxygen oxidoreductases , EC1.10.3.2) can oxidize various substrates, and those which are tolerant to and even activated by salts have attracted a lot of attention due to their application potential in certain industries. The mechanism of the salt activation of laccases is awaiting to be elucidated yet. Our previous study (Li, Xie et al. 2018) supposed that the salt activation of marine laccase Lac15 might be attributed to Cl - ion specifically binding to some local sites to interfere substrate binding and/or electron transfer . In this study, we found two sites whose mutations resulted in elimination of the salt activation of Lac15’s activity towards catechol and dopamine respectively, and revealed that the mutations affected the activity by altering both E m and k cat , demonstrating the supposed mechanism. A model for the salt activation of laccases was accordingly proposed, albeit some details are to be elucidated.
Li Jie,Xie Yanan,Wang Rui,Fang Zemin,Fang Wei,Zhang Xuecheng,Xiao Yazhong
Laccase (benzenediol: oxygen oxidoreductases, EC1.10.3.2) is a multi-copper oxidase capable of oxidizing a variety of phenolic and other aromatic organic compounds. The catalytic power of laccase makes it an attractive candidate for potential applications in many areas of industry including biodegradation of organic pollutants and synthesis of novel drugs. Most laccases are vulnerable to high salt and have limited applications. However, some laccases are not only tolerant to but also activated by certain concentrations of salt and thus have great application potential. The mechanisms of salt-induced activity enhancement of laccases are unclear as yet. In this study, we used dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, intrinsic fluorescence emission, circular dichroism, ultraviolet–visible light absorption, and an enzymatic assay to investigate the potential correlation between the structure and activity of the marine-derived laccase, Lac15, whose activity is promoted by low concentrations of NaCl. The results showed that low concentrations of NaCl exert little influence on the protein structure, which was partially folded in the absence of the salt; moreover, the partially folded rather than the fully folded state seemed to be favorable for enzyme activity, and this partially folded state was distinctive from the so-called ‘molten globule’ occasionally observed in active enzymes. More data indicated that salt might promote laccase activity through mechanisms involving perturbation of specific local sites rather than a change in global structure. Potential binding sites for chloride ions and their roles in enzyme activity promotion are proposed.
Laccases (benzenediol: oxygen oxidoreductases , EC1.10.3.2) can oxidize wide range of compounds thus have great application potential in diverse industries. The catalytic mechanisms of laccases have been extensively studied, while the details of proton transfer remain to be fully elucidated. In this study, we tried to uncover the sites that are crucial for the proton transfer of microbial laccase Lac15. A residue near the trinuclear copper center, D396, was indicated by statistical coupling analysis (SCA) and structural alignment to be an important site like D93, which is conserved in laccases and believed crucial for the catalysis by facilitating proton transfer. A representative mutant at this site, D396A, similar to D93A, exhibited significantly impaired catalysis with the global structure and substrate binding slightly perturbed. The mutation resulted in stay of the intermediate I, which would accept a proton to proceed to next catalysis stage, suggesting D396 might play a critical role in the proton transfer. Our finding may help to completely elucidate the proton transfer mechanism in laccases.
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