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

Osipov E, Polyakov K, Kittl R, Shleev S, Dorovatovsky P, Tikhonova T, Hann S, Ludwig R, Popov V - Acta Crystallogr. D Biol. Crystallogr. (2014)

Bottom Line: 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.Both structures did not contain the T2 copper ion.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: A. N. Bach Institute of Biochemistry, Leninsky Prospect 33/2, Moscow 119071, Russian Federation.

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.

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Glycosylation of BaL. (a) The carbohydrate chain attached to Asn194; (b) the carbohydrate chain attached to Asn82; (c) the α-mannose residue attached to Ser338. The carbohydrate chains are shown as ball-and-stick models. The protein surface is represented by grey balls (the Phe174 and Phe536 residues and the NAG residue attached to Asn39 are in cyan). The 2Fo − Fc electron-density maps (at the 1σ level) are shown in magenta. Hydrogen bonds are shown as black lines.
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fig3: Glycosylation of BaL. (a) The carbohydrate chain attached to Asn194; (b) the carbohydrate chain attached to Asn82; (c) the α-mannose residue attached to Ser338. The carbohydrate chains are shown as ball-and-stick models. The protein surface is represented by grey balls (the Phe174 and Phe536 residues and the NAG residue attached to Asn39 are in cyan). The 2Fo − Fc electron-density maps (at the 1σ level) are shown in magenta. Hydrogen bonds are shown as black lines.

Mentions: In the structure of BaL the carbohydrate chain at Asn194 contains six carbohydrate residues: MAN(α1→2)MAN(α1→6)MAN(α1→6)MAN(β1→4)NAG(β1→4)NAG(β1→Nδ)Asn194 (Fig. 3 ▶a). The terminal α-MAN residue (MAN557) in this chain forms two hydrogen bonds (MAN O6⋯Tyr33 OH and MAN O4⋯Glu32 N). This results in the stabilization of the orientation of this residue and can hinder its cleavage by α-mannosidase. The resistance of the NAG(β1→4)NAG bond in this chain to deglycosylation is attributed to the following factors: the location of this chain in the cavity on the protein surface and the stabilization of its conformation through hydrogen bonding between the terminal α-MAN residue and the first NAG residue (NAG O3⋯Val160 O). Owing to the stabilization of the conformation of the carbohydrate, the B factors of the terminal carbohydrate residues are much lower than those of the central residues. In the L499M BaL structure, the corresponding carbohydrate chain is shorter and only three residues [MAN(β1→4)NAG(β1→4)NAG(β1→Nδ)Asn194] were located in the electron-density map.


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

Osipov E, Polyakov K, Kittl R, Shleev S, Dorovatovsky P, Tikhonova T, Hann S, Ludwig R, Popov V - Acta Crystallogr. D Biol. Crystallogr. (2014)

Glycosylation of BaL. (a) The carbohydrate chain attached to Asn194; (b) the carbohydrate chain attached to Asn82; (c) the α-mannose residue attached to Ser338. The carbohydrate chains are shown as ball-and-stick models. The protein surface is represented by grey balls (the Phe174 and Phe536 residues and the NAG residue attached to Asn39 are in cyan). The 2Fo − Fc electron-density maps (at the 1σ level) are shown in magenta. Hydrogen bonds are shown as black lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4220974&req=5

fig3: Glycosylation of BaL. (a) The carbohydrate chain attached to Asn194; (b) the carbohydrate chain attached to Asn82; (c) the α-mannose residue attached to Ser338. The carbohydrate chains are shown as ball-and-stick models. The protein surface is represented by grey balls (the Phe174 and Phe536 residues and the NAG residue attached to Asn39 are in cyan). The 2Fo − Fc electron-density maps (at the 1σ level) are shown in magenta. Hydrogen bonds are shown as black lines.
Mentions: In the structure of BaL the carbohydrate chain at Asn194 contains six carbohydrate residues: MAN(α1→2)MAN(α1→6)MAN(α1→6)MAN(β1→4)NAG(β1→4)NAG(β1→Nδ)Asn194 (Fig. 3 ▶a). The terminal α-MAN residue (MAN557) in this chain forms two hydrogen bonds (MAN O6⋯Tyr33 OH and MAN O4⋯Glu32 N). This results in the stabilization of the orientation of this residue and can hinder its cleavage by α-mannosidase. The resistance of the NAG(β1→4)NAG bond in this chain to deglycosylation is attributed to the following factors: the location of this chain in the cavity on the protein surface and the stabilization of its conformation through hydrogen bonding between the terminal α-MAN residue and the first NAG residue (NAG O3⋯Val160 O). Owing to the stabilization of the conformation of the carbohydrate, the B factors of the terminal carbohydrate residues are much lower than those of the central residues. In the L499M BaL structure, the corresponding carbohydrate chain is shorter and only three residues [MAN(β1→4)NAG(β1→4)NAG(β1→Nδ)Asn194] were located in the electron-density map.

Bottom Line: 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.Both structures did not contain the T2 copper ion.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: A. N. Bach Institute of Biochemistry, Leninsky Prospect 33/2, Moscow 119071, Russian Federation.

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.

Show MeSH
Related in: MedlinePlus