Information on EC 3.4.24.75 - lysostaphin

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.4.24.75
-
RECOMMENDED NAME
GeneOntology No.
lysostaphin
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Hydrolysis of the -Gly-/-Gly- bond in the pentaglycine inter-peptide link joining staphylococcal cell wall peptidoglycans
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY hide
9011-93-2
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain BR11
-
-
Manually annotated by BRENDA team
strain BR11
-
-
Manually annotated by BRENDA team
strain ATCC 14917
-
-
Manually annotated by BRENDA team
strain GG
-
-
Manually annotated by BRENDA team
strain GG
-
-
Manually annotated by BRENDA team
subspecies cremoris MG1363
-
-
Manually annotated by BRENDA team
EPK1
-
-
Manually annotated by BRENDA team
EPK1
-
-
Manually annotated by BRENDA team
NRRL B-2628
-
-
Manually annotated by BRENDA team
strain TNK1
-
-
Manually annotated by BRENDA team
strain K-6-WI
-
-
Manually annotated by BRENDA team
strain K-6-WI
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Elastin + H2O
Fragments of elastin
show the reaction diagram
-
-
-
-
-
L-lysine(N-epsilon-pentaglycine-N-(2-aminobenzoyl))-[N-(2,4-dinitrophenyl)ethylenediamine] + H2O
?
show the reaction diagram
-
-
-
-
?
L-lysine(N-epsilon-pentaglycyl-DABCYL)-EDANS + H2O
?
show the reaction diagram
-
-
-
-
?
MV11 + H2O
?
show the reaction diagram
-
developing a fluorescence resonance energy transfer substrate based on MV11
-
-
?
N-(2-aminobenzoyl)pentaglycine-[N-(2,4-dinitrophenyl)ethylenediamine] + H2O
?
show the reaction diagram
-
-
-
-
?
N-Acetylhexaglycine + H2O
?
show the reaction diagram
-
-
-
-
-
peptidoglycan + H2O
?
show the reaction diagram
Peptidoglycan + H2O
Fragments of peptidoglycan
show the reaction diagram
soluble elastin + H2O
?
show the reaction diagram
-
-
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
peptidoglycan + H2O
?
show the reaction diagram
Peptidoglycan + H2O
Fragments of peptidoglycan
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Na+
-
stimulation
NH4+
-
stimulation
Zinc
-
0.9 mol zinc per mol LytM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
ampicillin
-
-
Co2+
-
10 mM, 30% decrease of activity of enzyme produced with pET vector system and 20% decrease of activity of enzyme produced with pBAD vector system
diammonium sulfate
-
-
diethyldicarbonate
iodoacetate
-
-
Ni2+
-
2 mM, 60% decrease of activity of enzyme produced with pET vector system and 20% decrease of activity of enzyme produced with pBAD vector system
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
C-terminus of TRAP
-
i.e. target of RNAIII activating protein, a membrane-associated protein in Staphylococcus sp., can enhance the activity of lysozyme and lysostaphin, interaction analysis, overview
-
additional information
-
the enzyme acts synergistically with oxacillin and with vancomycin in lysis of the cell wall of pathogenic Staphylococcus aureus
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.07
L-lysine(N-e-pentaglycine-N-(2-aminobenzoyl))-[N-(2,4-dinitrophenyl)ethylenediamine]
0.08
L-lysine(N-e-pentaglycyl-DABCYL)-EDANS
-
value from Grafit plot analysis
0.2 - 0.3
N-(2-Aminobenzoyl)pentaglycine-[N-(2,4-dinitrophenyl)ethylenediamine]
additional information
additional information
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
-
highest specific activity (turbidity reduction assay)
8
-
enzyme obtained from pET vector system
additional information
-
the pH value near a negatively charged cell is supposed to be strongly shifted to acidity as compared to the pH of the solution volume. This shifts the enzyme pH dependence curve in solution to alkalinity, overview
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
-
half-life of the staphylolytic activity approximately 2 months
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
stable for 72 h, recombinant enzyme
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9.5
-
calculated from amino acid sequence
9.7
-
mature enzyme
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Staphylococcus aureus (strain NCTC 8325)
Staphylococcus aureus (strain NCTC 8325)
Staphylococcus aureus (strain NCTC 8325)
Staphylococcus aureus (strain NCTC 8325)
Staphylococcus aureus (strain NCTC 8325)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
16260
-
ESI-TOF MS, endopeptidase domain (residues 1-140)
18400
-
N-terminal domain of gene product 13, 159 amino acids, calculated mass
22300
-
C-terminal domain of gene product 13, 202 amino acids, calculated mass
24500 - 27800
-
sedimentation equilibrium, sedimentation and diffusion, gel filtration, polyacylamide electrophoresis
26800
-
246 amino acids, mature peptide, recombinant enzyme
26900
-
calculated from the plot of mobility versus log molecular weight of standards
26920
-
mature lysostaphin, calculation from nucleotide sequence
27200
-
theoretically estimated value
28010
-
ESI-TOF MS, residues 247-493
29000
-
Sephacryl S-200 gel filtration, His6-fused lysostaphin
35600
-
mature enzyme, calculation from nucleotide sequence
39310
-
proenzyme, calculation from nucleotide sequence
40900
-
gp13 wild-type, 365 amino acids, calculated mass
42000
-
preproenzyme
42210
-
preprolysostaphin, calculation from nucleotide sequence
48000
-
SDS-PAGE in presence and absence of reducing agents
49120
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prolysostaphin, calculation from nucleotide sequence. Prolysostaphin is processed to the mature enzyme by an extracellular cysteine protease
64000 - 68000
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
-
C-terminal and N-terminal domain
monomer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
-
Asn125 is the exact glycosylation site of lysostaphin expressed in CMEC-08-D cells. N-glycosylation at Asn125 of lysostaphin decreases lysostaphin bacteriolytic activity but does not affect its binding ability to Staphylococcus aureus
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Computational modeling of the gene product 13-muropeptide complex for analysing the orientation of the scissile bond
-
sitting drop vapor diffusion method, using 0.1 M MES/NaOH pH 6.5 and 1.6 M magnesium sulfate, 2 mM tetraglycine phosphinic acid, or 0.1 M Tris (base), bicine pH 8.5, 0.1 M amino acids [L-Na-glutamate, L-alanine (racemic), glycine, L-lysine (racemic), L-serine (racemic)], 30% (v/v) PEG 8000, or 0.05 M potassium dihydrogen phosphate and 20% (w/v) PEG 8000
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
-
half-life at 5C 2 months
31508
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
-
half-life at pH 4 is 2 months
20 - 30
-
stable for 24 hours at pH 8.0
40
-
stable for 4h with 80% activity
60
-
15 min, marked decrease of activity
75
-
15 min, complete inactivation
100
-
15 min, stable. Activity is destroyed after 30 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
lysostaphin degrades Staphylococcus aureus cell walls inefficiently in low conductivity buffers (10-50 mS/cm)
lysostaphin is relatively stable when conjugated with polyethylene glycol and it maintains its activity in human serum
-
relatively unaffected by salt concentrations between 200 and 500 mM, LysK has a higher specific activity than lysostaphin at NaCl concentrations higher than 150 mM (turbidity reduction assay)
-
trypsin inactivates
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
n-Butanol
Pyridine
tert-Butanol
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
5C, 1 mM acetate buffer pH 4, half-life 2 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation and ion exchange chromatography
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Capto MMC column chromatography, and Sephadex G-50 gel filtration
-
cobalt Sepharose column chromatography
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from commercial preparation
-
immobilized metal affinity chromatography on Ni-NTA resin
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lysostaphin can be purified from Staphylococcus simulans strains
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metal-affinity chromatography and cation exchange chromatography
-
method not mentioned
-
recombinant enzyme from Escherichia coli
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recombinant His-tagged lysostaphin comprising residues 1 to 154 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
-
recombinant lysostaphin from Escherichia coli to homogeneity
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SP Sepharose column chromatography and Toyopearl PPG-600M column chromatography
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zinc metal-chelate chromatography, cation-exchange chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
amplified from pRG5 recombinant plasmid and cloned into Escherichia coli expression pTYB12 vector which allows the overexpression of a target protein as a fusion to a self-cleavabe affinity tag
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expressed as mature lysostaphin free from preprolysostaphin and prolysostaphin in Escherichia coli
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expressed in CMEC-08-D cells
-
expressed in Escherichia coli
-
expressed in Escherichia coli and Lactococcus lactis
expressed in Escherichia coli BL21 cells
-
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli M15 cells
-
expressed in Escherichia coli strain BL21(DE3)
-
expressed in HeLa cells
-
expression in Bacillus sphaericus
-
expression in Escherichia coli
-
expression in Escherichia coli with pUC19 as vector
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expression in Staphylococcus simulans, Staphylococcus aureus
-
expression of His-tagged lysostaphin comprising residues 1 to 154 in Escherichia coli strain BL21(DE3)
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expression of the functional mature enzyme fused to secretion signals of the Sep protein from Lactobacillus fermentum BR11, to direct the enzyme secretion from recombinant cells, in Lactobacillus fermentum strain BR11, Lactobacillus rhamnosus strain GG, Lactobacillus plantarum ATCC 14917 and Lactococcus lactis strain MG1363, method development and production levels, overview
-
identification of host factors in Lactococcus lactis subspecies cremoris MG1363 that affect the secretion efficiency of the peptidoglycan hydrolase lysostaphin by isolation and characterization of mutants generated by random transposon mutagenesis that had altered lysostaphin activity. Mutants with higher lysostaphin activity contain insertions in 4 different genes
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overexpression in Escherichia coli
-
recombinant expression in Escherichia coli
-
recominant expression of His-tagged lysostaphin by Escherichia coli BL21 (DE3) pEA3
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D195A
-
mutant of gene product 13, reduced general protein folding and Zn2+ binding (circular dichroism and particle-induced X-ray emission spectroscopy)
D195C
-
mutant of gene product 13, general protein folding and Zn2+ binding are maintained (circular dichroism and particle-induced X-ray emission spectroscopy)
D339C
-
the mutant shows about 37% of wild type bacteriolytic activity
D37A
-
no endopeptidase activity
H114A
-
no endopeptidase activity
H116A
-
no endopeptidase activity
H33A
-
no endopeptidase activity
H83A
-
no endopeptidase activity
K332C
-
the mutant shows about 82% of wild type bacteriolytic activity
K332C/T464C
-
the mutant retains around 25% of the bacteriolytic activity of the wild type protein
K336C
-
the mutant shows about 90% of wild type bacteriolytic activity
L258C
-
the mutant shows about 35% of wild type bacteriolytic activity
N125Q
-
the mutant possesses staphylolytic activity
N232Q
-
the mutant lacks staphylolytic activity
N260C
-
the mutant shows about 80% of wild type bacteriolytic activity
Q256C
-
the mutant shows about 85% of wild type bacteriolytic activity
R470C
-
the mutant shows about 50% of wild type bacteriolytic activity
T107A
-
reduced endopeptidase activity
T464C
-
the mutant shows about 85% of wild type bacteriolytic activity
T477C
-
the mutant shows wild type bacteriolytic activity
T482C
-
the mutant shows wild type bacteriolytic activity
V487C
-
the mutant shows wild type bacteriolytic activity
Y81A
-
reduced endopeptidase activity
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
-
improvement of lipid extraction of staphylococcal cells
diagnostics
-
detection of Staphylococcus aureus
food industry
-
antistaphylococcal agent
medicine
pharmacology
-
lysostaphin is useful for treatment for systemic Staphylococcus aureus infection in a mouse model
additional information
-
antistaphylococcal agent in clinical laboratories
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