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Literature summary extracted from

  • Andberg, M.; Hakulinen, N.; Auer, S.; Saloheimo, M.; Koivula, A.; Rouvinen, J.; Kruus, K.
    Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure (2009), FEBS J., 276, 6285-6300.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.10.3.2 expression of wild-type and mutant enzymes in Trichoderma reesei and Saccharomyces cerevisiae, expression of mutant L559A in Saccharomyces cerevisiae. The yeast is not able to process MaL correctly, and the additional 14 amino acids are present in the protein. Therefore, expression is performed with another construct, pMS175, where mature MaL cDNA, with a stop codon, is introduced after the C-terminal processing site. Changes in the C-terminus of MaL cause major defects in protein production in both expression hosts Melanocarpus albomyces

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.10.3.2 purified recombinant mutant L559A, by vapor diffusion method at 20°C, 10 mg/ml protein mixed with 15% PMME 2000, 0.2 M ammonium sulfate, and 0.1 M sodium acetate, pH 4.5, microseeding using 13% PMME 2000 and an equilibrium time of 4 h,usage of 25% glycerol as cryoprotectant, X-ray diffraction structure determination and analysis at 2. A resolution by molecular replacement Melanocarpus albomyces

Protein Variants

EC Number Protein Variants Comment Organism
1.10.3.2 L559A the C-terminal mutation affects the trinuclear site geometry of the mutant enzyme, which also shows 3-4fold reduced activity compared to the wild-type enzyme Melanocarpus albomyces
1.10.3.2 additional information deletion of the last four amino acids dramatically affected the activity of the enzyme, as the deletion mutant delDSGL559 is practically inactive Melanocarpus albomyces

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.10.3.2 NaN3
-
 Melanocarpus albomyces

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.10.3.2 0.011
-
 2,6-dimethoxyphenol pH 4.5, 25°C, recombinant wild-type enzyme Melanocarpus albomyces
1.10.3.2 0.016
-
 2,6-dimethoxyphenol pH 4.5, 25°C, recombinant mutant L559A expressed in Saccharomyces cerevisiae Melanocarpus albomyces
1.10.3.2 0.031
-
 syringaldazine pH 4.5, 25°C, recombinant mutant L559A expressed in Saccharomyces cerevisiae Melanocarpus albomyces
1.10.3.2 0.037
-
 syringaldazine pH 4.5, 25°C, recombinant wild-type enzyme Melanocarpus albomyces
1.10.3.2 0.26
-
 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) pH 4.5, 22°C, recombinant enzyme expressed in Saccharomyces cerevisiae Melanocarpus albomyces
1.10.3.2 0.28
-
 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) pH 4.5, 25°C, recombinant enzyme expressed in Trichoderma reesei Melanocarpus albomyces
1.10.3.2 0.4
-
 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) pH 4.5, 25°C, recombinant wild-type enzyme Melanocarpus albomyces
1.10.3.2 0.9
-
 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) pH 4.5, 25°C, recombinant mutant L559A expressed in Saccharomyces cerevisiae Melanocarpus albomyces

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.10.3.2 Cu2+ a copper-containing metalloenzyme Melanocarpus albomyces

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.10.3.2 70000
-
 x * 70000, recombinant nonglycosylated enzyme expressed in Trichodrma reesei, SDS-PAGE, x * 100000, recombinant glycosylated enzyme expressed in Saccharomyces cerevisiae, SDS-PAGE Melanocarpus albomyces
1.10.3.2 100000
-
 x * 70000, recombinant nonglycosylated enzyme expressed in Trichodrma reesei, SDS-PAGE, x * 100000, recombinant glycosylated enzyme expressed in Saccharomyces cerevisiae, SDS-PAGE Melanocarpus albomyces

Organism

EC Number Organism UniProt Comment Textmining
1.10.3.2 Melanocarpus albomyces Q70KY3
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
1.10.3.2 glycoprotein the recombinant MaL, expressed in Saccharomyces cerevisiae, is heavily overglycosylated, while the recombinant enzyme overexpressed in Trichoderma reesei is not Melanocarpus albomyces
1.10.3.2 proteolytic modification the C-terminus is post-translationally processed after Leu559, leading to removal of the last 14 aminoacids of the mature protein Melanocarpus albomyces

Purification (Commentary)

EC Number Purification (Comment) Organism
1.10.3.2 recombinant wild-type and mutant enzymes from Trichoderma reesei and Saccharomyces cerevisiae by two different steps of anion exchange chromatography, hydrophobic interaction chromatography, and gel filtration Melanocarpus albomyces

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.10.3.2 additional information
-
-
 Melanocarpus albomyces
1.10.3.2 24 36.6 recombinant enzyme expressed in Saccharomyces cerevisiae Melanocarpus albomyces
1.10.3.2 38.4 50.4 recombinant enzyme expressed in Trichoderma reesei Melanocarpus albomyces

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.10.3.2 2,2'-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid) + O2
-
 Melanocarpus albomyces ?
-
 ?
1.10.3.2 2,6-dimethoxyphenol + O2
-
 Melanocarpus albomyces ?
-
 ?
1.10.3.2 syringaldazine + O2
-
 Melanocarpus albomyces ?
-
 ?

Subunits

EC Number Subunits Comment Organism
1.10.3.2 ? x * 70000, recombinant nonglycosylated enzyme expressed in Trichodrma reesei, SDS-PAGE, x * 100000, recombinant glycosylated enzyme expressed in Saccharomyces cerevisiae, SDS-PAGE Melanocarpus albomyces
1.10.3.2 More in MaL, the four C-terminal amino acids of the mature protein penetrate into a tunnel leading towards the trinuclear site. The C-terminal carboxylate group forms a hydrogen bond with a side chain of His140, which also coordinates to the type 3 copper, three-dimensional structure of mutant L559A, overview Melanocarpus albomyces

Synonyms

EC Number Synonyms Comment Organism
1.10.3.2 MAL
-
 Melanocarpus albomyces
1.10.3.2 p-diphenol dioxygen oxidoreductase
-
 Melanocarpus albomyces

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.10.3.2 22 25 assay at Melanocarpus albomyces

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.10.3.2 40
-
 half-life of recombinant, expressed in Saccharomyces cerevisiae, wild-type enzyme is over 50 h, and of the L559A mutant is 24 h Melanocarpus albomyces
1.10.3.2 50
-
 half-life of recombinant, expressed in Saccharomyces cerevisiae, wild-type enzyme is 23 h, and of the L559A mutant is 6 h Melanocarpus albomyces
1.10.3.2 60
-
 half-life of recombinant, expressed in Saccharomyces cerevisiae, wild-type enzyme is 4.5 h, and of the L559A mutant is below 10 min Melanocarpus albomyces

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.10.3.2 6.57
-
 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) pH 4.5, 25°C, recombinant mutant L559A expressed in Saccharomyces cerevisiae Melanocarpus albomyces
1.10.3.2 9.08
-
 2,6-dimethoxyphenol pH 4.5, 25°C, recombinant mutant L559A expressed in Saccharomyces cerevisiae Melanocarpus albomyces
1.10.3.2 10.2
-
 2,6-dimethoxyphenol pH 4.5, 25°C, recombinant wild-type enzyme Melanocarpus albomyces
1.10.3.2 21.05
-
 syringaldazine pH 4.5, 25°C, recombinant mutant L559A expressed in Saccharomyces cerevisiae Melanocarpus albomyces
1.10.3.2 28.1
-
 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) pH 4.5, 25°C, recombinant wild-type enzyme Melanocarpus albomyces
1.10.3.2 40.17
-
 syringaldazine pH 4.5, 25°C, recombinant wild-type enzyme Melanocarpus albomyces

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.10.3.2 4
-
-
 Melanocarpus albomyces

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.10.3.2 3 8 activity range Melanocarpus albomyces

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
1.10.3.2 5.5 8 the L559A mutant remains stable within this pH range after 330 h at 4°C, the enzyme looses 60% at pH 5.0, and 95% at pH 4.0 after 330 h. No activity remains at pH 3.0 and pH 2.0 after 330 h Melanocarpus albomyces

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.10.3.2 0.0079
-
 NaN3 pH 4.5, 25°C, recombinant wild-type enzyme versus 2,2'-azinobis(3-ethylbenzo-6-thiazolinesulfonic) acid Melanocarpus albomyces
1.10.3.2 0.029
-
 NaN3 pH 4.5, 25°C, recombinant wild-type enzyme, versus 2,6-dimethoxyphenol Melanocarpus albomyces
1.10.3.2 0.055
-
 NaN3 pH 4.5, 25°C, recombinant mutant L559A expressed in Saccharomyces cerevisiae, versus 2,6-dimethoxyphenol Melanocarpus albomyces
1.10.3.2 0.085
-
 NaN3 pH 4.5, 25°C, recombinant mutant L559A expressed in Saccharomyces cerevisiae versus 2,2'-azinobis(3-ethylbenzo-6-thiazolinesulfonic) acid Melanocarpus albomyces

pI Value

EC Number Organism Comment pI Value Maximum pI Value
1.10.3.2 Melanocarpus albomyces recombinant MaL, expressed in Saccharomyces cerevisiae, has several pI forms, whereas the recombinant MaL, produced in Trichoderma reesei only had one pI form at pI 4.0
-
 3.55

General Information

EC Number General Information Comment Organism
1.10.3.2 malfunction changes in the C-terminus of MaL caused major defects in protein production in both expression hosts Melanocarpus albomyces