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Literature summary extracted from

  • Durao, P.; Chen, Z.; Silva, C.S.; Soares, C.M.; Pereira, M.M.; Todorovic, S.; Hildebrandt, P.; Bento, I.; Lindley, P.F.; Martins, L.O.
    Proximal mutations at the type 1 copper site of CotA laccase: spectroscopic, redox, kinetic and structural characterization of I494A and L386A mutants (2008), Biochem. J., 412, 339-346.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.10.3.2 expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) resulting in strains AH3547 and AH3560 Bacillus subtilis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.10.3.2 recombinant mutant enzymes I494A and L386A at 20°C and 10.8 mg/ml or 7.9 mg/ml, respectively, from a crystallization solution containing 0.1 M sodium citrate, 8-10% PEG MME 5000 and 14% propan-2-ol at pH 5.5, X-ray diffraction structure determination and analysis at 1.6-2.9 A resolution, cryoprotection of crystals of mutants I494A and L386A by 22% ethylene glycol or 25% glycerol, respectively Bacillus subtilis

Protein Variants

EC Number Protein Variants Comment Organism
1.10.3.2 I494A site-directed mutagenesis at a hydrophobic residue in the vicinity of the type 1 copper site, the replacement of Ile494 by an alanine residue leads to significant changes in the enzyme, the mutant shows differences in the type 1 as well as in the type 2 copper centre compared to the wild-type enzyme Bacillus subtilis
1.10.3.2 L386A the site-directed mutation of Leu386, a hydrophobic residue in the vicinity of the type 1 copper site, to an alanine residue appears to cause only very subtle alterations in the properties of the enzyme indicating minimal changes in the structure of the copper centres Bacillus subtilis
1.10.3.2 M502F replacement of Met502, which is weakly co-ordinating to the T1 copper, in CotA laccase by the non-co-ordinating residues leucine and phenylalanine allows the maintenance of the T1 copper geometry while causing an increase in the redox potential Bacillus subtilis
1.10.3.2 M502L replacement of Met502, which is weakly co-ordinating to the T1 copper, in CotA laccase by the non-co-ordinating residues leucine and phenylalanine allows the maintenance of the T1 copper geometry while causing an increase in the redox potential Bacillus subtilis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.10.3.2 0.018
-
 syringaldazine pH 7.0, 25°C, wild-type CotA Bacillus subtilis
1.10.3.2 0.033
-
 syringaldazine pH 7.0, 25°C, mutant L386A Bacillus subtilis
1.10.3.2 0.052
-
 syringaldazine pH 8.0, 25°C, mutant I494A Bacillus subtilis
1.10.3.2 0.124
-
 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) pH 4.0, 25°C, wild-type CotA Bacillus subtilis
1.10.3.2 0.145
-
 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) pH 4.0, 25°C, mutant L386A Bacillus subtilis
1.10.3.2 0.227
-
 2,6-dimethoxyphenol pH 7.0, 25°C, wild-type CotA Bacillus subtilis
1.10.3.2 0.576
-
 2,6-dimethoxyphenol pH 7.0, 25°C, mutant L386A Bacillus subtilis
1.10.3.2 1.295
-
 2,6-dimethoxyphenol pH 9.0, 25°C, mutant I494A Bacillus subtilis
1.10.3.2 2.027
-
 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) pH 4.0, 25°C, mutant I494A Bacillus subtilis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.10.3.2 Cu2+ a copper enzyme, the wild-type enzyme contains 3.7 mol Cu2+ per mol of enzyme, mutants I494A and L386A contain 4.0 mol copper per mol of enzyme. Binding structures in wild-type and mutant enzymes I494A and L386A, analysis of crystal structures and simulation, overview Bacillus subtilis

Organism

EC Number Organism UniProt Comment Textmining
1.10.3.2 Bacillus subtilis
-
 strains AH3517 containing pLOM10, AH3547 and AH3560
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.10.3.2 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) + O2
-
 Bacillus subtilis ?
-
 ?
1.10.3.2 2,6-dimethoxyphenol + O2
-
 Bacillus subtilis ?
-
 ?
1.10.3.2 syringaldazine + O2
-
 Bacillus subtilis ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.10.3.2 CotA
-
 Bacillus subtilis
1.10.3.2 More laccases are members of the MCO, multicopper oxidase, family of enzymes Bacillus subtilis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.10.3.2 25
-
 assay at Bacillus subtilis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.10.3.2 3 6 2,6-dimethoxyphenol pH 7.0, 25°C, wild-type CotA Bacillus subtilis
1.10.3.2 4.5
-
 2,6-dimethoxyphenol pH 9.0, 25°C, mutant I494A Bacillus subtilis
1.10.3.2 7.2
-
 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) pH 4.0, 25°C, mutant I494A Bacillus subtilis
1.10.3.2 9
-
 syringaldazine pH 8.0, 25°C, mutant I494A Bacillus subtilis
1.10.3.2 13
-
 syringaldazine pH 7.0, 25°C, mutant L386A Bacillus subtilis
1.10.3.2 17
-
 2,6-dimethoxyphenol pH 7.0, 25°C, mutant L386A Bacillus subtilis
1.10.3.2 52
-
 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) pH 4.0, 25°C, mutant L386A Bacillus subtilis
1.10.3.2 80
-
 syringaldazine pH 7.0, 25°C, wild-type CotA Bacillus subtilis
1.10.3.2 322
-
 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) pH 4.0, 25°C, wild-type CotA Bacillus subtilis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.10.3.2 4
-
 substrate 2,2'-azinobis(3-ethylbenzo-6-thiazolinesulfonic) acid, wild-type enzyme and mutant enzymes Bacillus subtilis
1.10.3.2 7
-
 substrate 2,6-dimethoxyphenol, assay at for the wild-type enzyme and the L386A mutant Bacillus subtilis
1.10.3.2 7.6
-
 redox titration assay at Bacillus subtilis
1.10.3.2 8 9 for the I494A mutant Bacillus subtilis

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.10.3.2 additional information
-
 pH profiles of wild-type and mutant enzymes Bacillus subtilis