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Literature summary for 3.4.24.20 extracted from

  • Oedum, A.S.; Oestergaard, S.; Noerby, I.; Meldal, M.; Olesen, K.
    Heterologous expression of peptidyl-Lys metallopeptidase of Armillaria mellea and mutagenic analysis of the recombinant peptidase (2016), J. Biochem., 159, 461-470 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.4.24.20

Cloned(Commentary)

Cloned (Comment) Organism
gene lysn, recombinant expression of C-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain strain BL21(DE3) or Pichia pastoris strain CBS 704, expression method evaluation Armillaria mellea

Protein Variants

Protein Variants Comment Organism
A101D site-directed mutagenesis, the mutant shows increased specificity towards arginine in subsites S2'-S4' compared to the wild-type protease Armillaria mellea
D132N site-directed mutagenesis, mutation in the zinc binding motif, leading to a significant decrease in activity, showing 16% of wild-type protease activity Armillaria mellea
D156A site-directed mutagenesis, 99% reduced activity compared to wild-type Armillaria mellea
E120Q site-directed mutagenesis, mutation in the zinc binding motif, 97% reduced activity compared to wild-type Armillaria mellea
G136L site-directed mutagenesis, 96% reduced activity compared to wild-type Armillaria mellea
H123R site-directed mutagenesis, mutation in the zinc binding motif, leading to 99% decrease in activity compared to wild-type Armillaria mellea
T105D site-directed mutagenesis, the mutant shows increased specificity towards arginine in subsites S2'-S4' compared to the wild-type protease Armillaria mellea
T130A site-directed mutagenesis, 96% reduced activity compared to wild-type Armillaria mellea
Y135F site-directed mutagenesis, 95% reduced activity compared to wild-type Armillaria mellea
Y135W site-directed mutagenesis, 95% reduced activity compared to wild-type Armillaria mellea
Y85L site-directed mutagenesis, 97% reduced activity compared to wild-type Armillaria mellea

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michealis-Menten equation by nonlinear regression Armillaria mellea
0.00037
-
 SAQKRVS recombinant wild-type enzyme, pH 7.5, temperature not specified in the publication Armillaria mellea
0.00076
-
 SAQKMRS recombinant wild-type enzyme, pH 7.5, temperature not specified in the publication Armillaria mellea
0.0025
-
 SAQKMVS recombinant wild-type enzyme, pH 7.5, temperature not specified in the publication Armillaria mellea
0.00393
-
 SAQKMVR recombinant wild-type enzyme, pH 7.5, temperature not specified in the publication Armillaria mellea

Metals/Ions

Metals/Ions Comment Organism Structure
Zn2+ enzyme Am-LysN has three zinc coordinating residues His119, His123 and Asp132 while two water molecules act as fourth and fifth zinc ligand Armillaria mellea

Organism

Organism UniProt Comment Textmining
Armillaria mellea Q9Y7F7
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain strain BL21(DE3) or Pichia pastoris strain CBS 704 Armillaria mellea

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information substrate specificity of wild-type and mutant enzymes, overview Armillaria mellea ?
-
 ?
SAQKFVS + H2O
-
 Armillaria mellea SAQ + KFVS
-
 ?
SAQKIVS + H2O
-
 Armillaria mellea SAQ + KIVS
-
 ?
SAQKLVS + H2O
-
 Armillaria mellea SAQ + KLVS
-
 ?
SAQKMFS + H2O
-
 Armillaria mellea SAQ + KMFS
-
 ?
SAQKMIS + H2O
-
 Armillaria mellea SAQ + KMIS
-
 ?
SAQKMLS + H2O
-
 Armillaria mellea SAQ + KMLS
-
 ?
SAQKMNS + H2O
-
 Armillaria mellea SAQ + KMNS
-
 ?
SAQKMRS + H2O
-
 Armillaria mellea SAQ + KMRS
-
 ?
SAQKMRVR + H2O
-
 Armillaria mellea SAQ + KMRVR
-
 ?
SAQKMRVS + H2O
-
 Armillaria mellea SAQ + KMRVS
-
 ?
SAQKMSS + H2O
-
 Armillaria mellea SAQ + KMSS
-
 ?
SAQKMVF + H2O
-
 Armillaria mellea SAQ + KMVF
-
 ?
SAQKMVL + H2O
-
 Armillaria mellea SAQ + KMVL
-
 ?
SAQKMVR + H2O
-
 Armillaria mellea SAQ + KMVR
-
 ?
SAQKMVS + H2O
-
 Armillaria mellea SAQ + KMVS
-
 ?
SAQKMVV + H2O
-
 Armillaria mellea SAQ + KMVV
-
 ?
SAQKNVS + H2O
-
 Armillaria mellea SAQ + KNVS
-
 ?
SAQKRVS + H2O
-
 Armillaria mellea SAQ + KRVS
-
 ?
SAQKSVS + H2O
-
 Armillaria mellea SAQ + KSVS
-
 ?
SAQKVVS + H2O
-
 Armillaria mellea SAQ + KVVS
-
 ?
SFQKMVS + H2O
-
 Armillaria mellea SFQ + KMVS
-
 ?
SLQKMVS + H2O
-
 Armillaria mellea SLQ + KMVS
-
 ?
SMQKMVS + H2O
-
 Armillaria mellea SMQ + KMVS
-
 ?
SNQKMVS + H2O
-
 Armillaria mellea SNQ + KMVS
-
 ?
SSQKMVS + H2O
-
 Armillaria mellea SSQ + KMVS
-
 ?
SVQKMVS + H2O
-
 Armillaria mellea SVQ + KMVS
-
 ?

Synonyms

Synonyms Comment Organism
Am-LysN
-
 Armillaria mellea
aspzincin metalloprotease
-
 Armillaria mellea
LysN
-
 Armillaria mellea
peptidyl-Lys metallopeptidase
-
 Armillaria mellea

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.26
-
 SAQKRVS recombinant wild-type enzyme, pH 7.5, temperature not specified in the publication Armillaria mellea
0.64
-
 SAQKMRS recombinant wild-type enzyme, pH 7.5, temperature not specified in the publication Armillaria mellea
1
-
 SAQKMVS recombinant wild-type enzyme, pH 7.5, temperature not specified in the publication Armillaria mellea
1.51
-
 SAQKMVR recombinant wild-type enzyme, pH 7.5, temperature not specified in the publication Armillaria mellea

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Armillaria mellea

General Information

General Information Comment Organism
malfunction mutants with extended S1' binding pocket show specificity towards arginine in subsites S2'-S4' compared to the wild-type protease resulting from the additional negative charge which attract and interact with positively charged residues better than the wild-type. The strict lysine specificity at P1' is preserved in the recombinant rAm-LysN. Relative specificity of site-directed mutants in the substrate binding sites of rAm-LysN compared to the wild-type enzyme with the reference peptide SAQKMVS, overview Armillaria mellea
additional information identification of catalytic residues (Y85, E120, H123, T130, D132, Y135, G136, and D156) and mechanism of work, overview. The S1' binding pocket of LysN is quite narrow and lined with negative charge to specifically accommodate lysine. Residues M65, D83, V88, A101, T105, Q131, Q137, and E159 are involved in substrate specificity. Homology model of Am-LysN based on the crystal structure of Gf-LysN, molecular dynamic simulations, proposed subsite interaction, structure-function analysis, overview Armillaria mellea
physiological function peptidyl-Lys metallopeptidase (LysN) from Armillaria mellea is an aspzincin metalloprotease, which cleaves in front of lysines Armillaria mellea

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
207.7
-
 SAQKRVS recombinant wild-type enzyme, pH 7.5, temperature not specified in the publication Armillaria mellea
384.2
-
 SAQKMVR recombinant wild-type enzyme, pH 7.5, temperature not specified in the publication Armillaria mellea
400
-
 SAQKMVS recombinant wild-type enzyme, pH 7.5, temperature not specified in the publication Armillaria mellea
842.1
-
 SAQKMRS recombinant wild-type enzyme, pH 7.5, temperature not specified in the publication Armillaria mellea