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Effect of the L499M mutation of the ascomycetous Botrytis aclada laccase on redox potential and catalytic properties.

ABSTRACT: 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: E0 = 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.

SUBMITTER: Osipov E

PROVIDER: S-EPMC4220974 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Effect of the L499M mutation of the ascomycetous Botrytis aclada laccase on redox potential and catalytic properties.

Osipov Evgeny E Polyakov Konstantin K Kittl Roman R Shleev Sergey S Dorovatovsky Pavel P Tikhonova Tamara T Hann Stephan S Ludwig Roland R Popov Vladimir V

Acta crystallographica. Section D, Biological crystallography 20141023 Pt 11

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 ...[more]

PMID: 25372682

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Project description:A new monoiminoacenaphthenone 3,5-(CF3)2C6H3-mian (complex 2) was synthesized and further exploited, along with the already known monoiminoacenaphthenone dpp-mian, to obtain oxidovanadium(IV) complexes [VOCl2(dpp-mian)(CH3CN)] (3) and [VOCl(3,5-(CF3)2C6H3-bian)(H2O)][VOCl3(3,5-(CF3)2C6H3-bian)]·2.85DME (4) from [VOCl2(CH3CN)2(H2O)] (1) or [VCl3(THF)3]. The structure of all compounds was determined using X-ray structural analysis. The vanadium atom in these structures has an octahedral coordination environment. Complex 4 has an unexpected structure. Firstly, it contains 3,5-(CF3)2C6H3-bian instead of 3,5-(CF3)2C6H3-mian. Secondly, it has a binuclear structure, in contrast to 3, in which two oxovanadium parts are linked to each other through V=O···V interaction. This interaction is non-covalent in origin, according to DFT calculations. In structures 2 and 3, non-covalent π-π staking interactions between acenaphthene moieties of the neighboring molecules (distances are 3.36-3.40 Å) with an estimated energy of 3 kcal/mol were also found. The redox properties of the obtained compounds were studied using cyclic voltammetry in solution. In all cases, the reduction processes initiated by the redox-active nature of the mian or bian ligand were identified. The paramagnetic nature of complexes 3 and 4 has been proven by EPR spectroscopy. Complexes 3 and 4 exhibited high catalytic activity in the oxidation of alkanes and alcohols with peroxides. The yields of products of cyclohexane oxidation were 43% (complex 3) and 27% (complex 4). Based on the data regarding the study of regio- and bond-selectivity, it was concluded that hydroxyl radicals play the most crucial role in the reaction. The initial products in the reactions with alkanes are alkyl hydroperoxides, which are easily reduced to their corresponding alcohols by the action of triphenylphosphine (PPh3). According to the DFT calculations, the difference in the catalytic activity of 3 and 4 is most likely associated with a different mechanism for the generation of ●OH radicals. For complex 4 with electron-withdrawing CF3 substituents at the diimine ligand, an alternative mechanism, different from Fenton's and involving a redox-active ligand, is assumed.

Dataset Information

Effect of the L499M mutation of the ascomycetous Botrytis aclada laccase on redox potential and catalytic properties.

Publications

Effect of the L499M mutation of the ascomycetous Botrytis aclada laccase on redox potential and catalytic properties.

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