title: Insight into stability of CotA laccase from the spore coat of Bacillus subtilis.
abstract: The axial ligand of the catalytic mononuclear T1 copper site (Met(502)) of the CotA laccase was replaced by a leucine or phenylalanine residue to increase the redox potential of the enzyme. These mutations led to an increase in the redox potential by approx. 100 mV relative to the wild-type enzyme but the catalytic constant k(cat) in the mutant enzymes was severely compromised. This decrease in the catalytic efficiency was unexpected as the X-ray analysis of mutants has shown that replacement of methionine ligand did not lead to major structural changes in the geometry of the T1 centre or in the overall fold of the enzyme. However, the mutations have a profound impact on the thermodynamic stability of the enzyme. The fold of the enzyme has become unstable especially with the introduction of the larger phenylalanine residue and this instability should be related to the decrease in the catalytic efficiency. The instability of the fold for the mutant proteins resulted in the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states. Quenching of tryptophan fluorescence by acrylamide has further revealed that the intermediate state is partly unfolded.
authors.affiliation: Instituto de Biotecnologia e Bioengenharia, Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.
authors.forename: E P
authors.initials: EP
authors.lastname: Melo
authors.forename: A T
authors.initials: AT
authors.lastname: Fernandes
authors.forename: P
authors.initials: P
authors.lastname: Durão
authors.forename: L O
authors.initials: LO
authors.lastname: Martins
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D004795:Enzyme Stability; D042845:Laccase / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013171:Spores, Bacterial / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D042845:Laccase