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 (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 | 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 | Comment | Organism | Structure |
---|---|---|---|
NaN3 | - |
Melanocarpus albomyces |
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 | Comment | Organism | Structure |
---|---|---|---|
Cu2+ | a copper-containing metalloenzyme | Melanocarpus albomyces |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Melanocarpus albomyces | Q70KY3 | - |
- |
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 (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 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 | 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 | 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 | Comment | Organism |
---|---|---|
MAL | - |
Melanocarpus albomyces |
p-diphenol dioxygen oxidoreductase | - |
Melanocarpus albomyces |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | 25 | assay at | Melanocarpus albomyces |
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 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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
4 | - |
- |
Melanocarpus albomyces |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
3 | 8 | activity range | Melanocarpus albomyces |
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 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 |
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 | Comment | Organism |
---|---|---|
malfunction | changes in the C-terminus of MaL caused major defects in protein production in both expression hosts | Melanocarpus albomyces |