Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis: structural, biochemical, enzymatic and stability studies
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Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme (E 0=455 mV). However the M502L mutant exhibits a twofold to fourfold decrease in the k cat values for the all substrates tested and the catalytic activity in M502F is even more severely compromised; 10% activity and 0.15–0.05% for the non-phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity.
KeywordsLaccases T1 Cu site Site-directed mutagenesis Redox potential Enzyme stability
We would like to thank our colleagues at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB-UNL), Cláudio M. Soares, António M. Baptista and Manuela M. Pereira for support and useful discussions. We would also like to thank João Carita for help with the cell growth in the Organic Fermentation Unit at the ITQB-UNL. The ITQB-UNL and the Fundação para a Ciência e a Tecnológia provided the resources necessary for this research. All X-ray data were collected at the European Synchrotron Radiation Facility, Grenoble, France, with the kind assistance of the scientists responsible for the operation of beam line ID29.
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