Insight into stability of CotA laccase from the spore coat of Bacillus subtilis

E.P Melo,A.T Fernandes,L.O Martins,P Durão

doi:10.1042/bst0351579

E.P Melo, A.T Fernandes + Show 2 more

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https://doi.org/10.1042/bst0351579

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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.

Highlights

Laccases are members of the multicopper oxidase family of enzymes that includes ascorbate oxidase and ceruloplasmin [1,2]
The role of the T1 site within the multicopper oxidases is related to the long-range intramolecular electron transfer, shuttling the electrons from the reduced substrate to the trinuclear centre, where O2 is reduced to water
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

Summary

Introduction

Laccases are members of the multicopper oxidase family of enzymes that includes ascorbate oxidase and ceruloplasmin [1,2]. Fungal laccases have a non-co-ordinating phenylalanine or leucine residue at the axial position and these may contribute, at least in part, to the high redox potential observed in these enzymes. To test this hypothesis, we have performed site-directed mutagenesis of the methionine residue in the axial position of the T1 copper site of CotA (position 502) to leucine and phenylalanine residues and analysed the effect of these mutations on the kinetics, redox potential and stability of the enzyme [9]. The site-directed replacement of Met502, an axial ligand of the T1 copper site, by the non-co-ordinating residue leucine and phenylalanine resulted in enzymes with the characteristic fold of the wild-type protein [9]. The slight movement of the mutated residue towards the protein surface, and away from the T1 copper atom, leads to a concerted movement of this region, pushing it away towards the solvent and slightly increasing the exposure

C The Authors Journal compilation C 2007 Biochemical Society

Findings

Concluding remarks