Effect of the L499M mutation of the ascomycetous Botrytis aclada laccase on redox potential and catalytic properties
- A. N. Bach Institute of Biochemistry, Leninsky Prospect 33/2, Moscow 119071 (Russian Federation)
- BOKU – University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Wien (Austria)
- RSC ‘Kurchatov Institute’, Acad. Kurchatov Sq. 1, Moscow 123182 (Russian Federation)
The structures of the ascomycetous B. aclada laccase and its L499M T1-site mutant have been solved at 1.7 Å resolution. The mutant enzyme shows a 140 mV lower redox potential of the type 1 copper and altered kinetic behaviour. The wild type and the mutant have very similar structures, which makes it possible to relate the changes in the redox potential to the L499M mutation Laccases are members of a large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. These enzymes contain four Cu atoms per molecule organized into three sites: T1, T2 and T3. In all laccases, the T1 copper ion is coordinated by two histidines and one cysteine in the equatorial plane and is covered by the side chains of hydrophobic residues in the axial positions. The redox potential of the T1 copper ion influences the enzymatic reaction and is determined by the nature of the axial ligands and the structure of the second coordination sphere. In this work, the laccase from the ascomycete Botrytis aclada was studied, which contains conserved Ile491 and nonconserved Leu499 residues in the axial positions. The three-dimensional structures of the wild-type enzyme and the L499M mutant were determined by X-ray crystallography at 1.7 Å resolution. Crystals suitable for X-ray analysis could only be grown after deglycosylation. Both structures did not contain the T2 copper ion. The catalytic properties of the enzyme were characterized and the redox potentials of both enzyme forms were determined: E{sub 0} = 720 and 580 mV for the wild-type enzyme and the mutant, respectively. Since the structures of the wild-type and mutant forms are very similar, the change in the redox potential can be related to the L499M mutation in the T1 site of the enzyme.
- OSTI ID:
- 22347727
- Journal Information:
- Acta Crystallographica. Section D: Biological Crystallography, Vol. 70, Issue Pt 11; Other Information: PMCID: PMC4220974; PMID: 25372682; PUBLISHER-ID: lv5066; OAI: oai:pubmedcentral.nih.gov:4220974; Copyright (c) Osipov et al. 2014; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0907-4449
- Country of Publication:
- Denmark
- Language:
- English
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