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Literature summary for 1.10.3.2 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)

Cloned (Comment) Organism
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)

Crystallization (Comment) Organism
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

Protein Variants Comment Organism
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
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

Inhibitors Comment Organism Structure
NaN3
-
Melanocarpus albomyces

KM Value [mM]

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

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

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
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
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

Organism UniProt Comment Textmining
Melanocarpus albomyces Q70KY3
-
-

Posttranslational Modification

Posttranslational Modification Comment Organism
glycoprotein the recombinant MaL, expressed in Saccharomyces cerevisiae, is heavily overglycosylated, while the recombinant enzyme overexpressed in Trichoderma reesei is not Melanocarpus albomyces
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)

Purification (Comment) Organism
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]

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

Substrates and Products (Substrate)

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

Subunits

Subunits Comment Organism
? x * 70000, recombinant nonglycosylated enzyme expressed in Trichodrma reesei, SDS-PAGE, x * 100000, recombinant glycosylated enzyme expressed in Saccharomyces cerevisiae, SDS-PAGE Melanocarpus albomyces
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

Synonyms Comment Organism
MAL
-
Melanocarpus albomyces
p-diphenol dioxygen oxidoreductase
-
Melanocarpus albomyces

Temperature Optimum [°C]

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

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
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
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
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]

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

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
4
-
-
Melanocarpus albomyces

pH Range

pH Minimum pH Maximum Comment Organism
3 8 activity range Melanocarpus albomyces

pH Stability

pH Stability pH Stability Maximum Comment Organism
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]

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

Organism Comment pI Value Maximum pI Value
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

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