Information on EC 3.4.24.28 - bacillolysin

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The expected taxonomic range for this enzyme is: Firmicutes

EC NUMBER
COMMENTARY
3.4.24.28
-
RECOMMENDED NAME
GeneOntology No.
bacillolysin
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
similar, but not identical, to that of thermolysin
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
hydrolysis of peptide bond
-
-
-
-
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Anilozyme P 10
-
-
-
-
Bacillus metalloendopeptidase
-
-
-
-
Bacillus metalloproteinase
-
-
-
-
Bacillus neutral proteinase
-
-
-
-
Bacillus polymyxa protease
-
-
Bacillus subtilis neutral proteinase
-
-
-
-
BL-MA
Bacillus megaterium A9542
-
-
-
C histolyticum neutral protease
-
-
EC 3.4.24.4
-
-
formerly
-
MCP 76
-
-
-
-
Megateriopeptidase
-
-
-
-
MprBi
B3V4Z0
-
Neutral protease
-
-
-
-
Neutral protease
-
-
Neutral protease
-
-
Neutral protease
-
-
non-specific neutral protease
-
-
Thermostable neutral protease
-
-
-
-
thermoysin-like proteinase
-
-
CAS REGISTRY NUMBER
COMMENTARY
76774-43-1
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
gene mprBi
UniProt
Manually annotated by BRENDA team
Bacillus megaterium A9542
A9542
-
-
Manually annotated by BRENDA team
strain 76
-
-
Manually annotated by BRENDA team
Bacillus pumilus 76
strain 76
-
-
Manually annotated by BRENDA team
var. amylosacchariticus
-
-
Manually annotated by BRENDA team
NCIB 8924 and NRRL B-3880
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
evolution
B3V4Z0, -
MprBi is a metzincin clan adamalysin/reprolysin-like metalloprotease and belongs to the class of zinc-dependent metalloproteinases
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-aminobenzoyl-AGLA-4-nitrobenzylamide + H2O
?
show the reaction diagram
-
-
-
-
?
2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide + H2O
?
show the reaction diagram
-
-
-
-
?
2-Furanacryloyl-Gly-OLeu-NH2 + H2O
?
show the reaction diagram
-
-
-
-
-
3-(2-furylacryloyl)-glycyl-L-leucinamide + H2O
?
show the reaction diagram
-
-
-
-
?
3-(2-furylacryloyl)-glycyl-L-leucine amide + H2O
?
show the reaction diagram
-
-
-
?
3-(2-furylacryloyl)-glycyl-L-leucine amide + H2O
?
show the reaction diagram
Bacillus megaterium, Bacillus megaterium A9542
-
-
-
-
-
3-(2-furylacryloyl)-L-alanyl-L-phenylalanine amide + H2O
?
show the reaction diagram
-
-
-
?
azocasein + H2O
?
show the reaction diagram
B3V4Z0, -
-
-
-
?
Benzoyl-Gly-OLeu-Ala + H2O
?
show the reaction diagram
-
-
-
-
-
Benzoyl-Gly-OPhe-Ala + H2O
?
show the reaction diagram
-
-
-
-
-
beta-casein + H2O
hydrolyzed casein
show the reaction diagram
-
-
-
?
Bovine serum albumin + H2O
?
show the reaction diagram
-
8.7% of the activity with denatured casein
-
-
?
casein + H2O
?
show the reaction diagram
-
-
-
-
?
casein + H2O
?
show the reaction diagram
-
-
-
-
-
casein + H2O
?
show the reaction diagram
P43133
-
-
-
?
casein + H2O
?
show the reaction diagram
Bacillus megaterium A9542
-
-
-
-
?
coagulation factor X + H2O
?
show the reaction diagram
Bacillus megaterium, Bacillus megaterium A9542
-
-
-
-
?
Collagen + H2O
?
show the reaction diagram
-
1.3% of the activity with denatured casein
-
-
?
Denatured casein + H2O
?
show the reaction diagram
-
-
-
-
?
denatured collagen + H2O
?
show the reaction diagram
-
11.5% of the activity with denatured casein
-
-
?
Dnp-AALR-NH2 + H2O
?
show the reaction diagram
B3V4Z0, -
-
-
-
?
Dnp-AAVR + H2O
?
show the reaction diagram
B3V4Z0, -
-
-
-
?
Dnp-GGFR + H2O
?
show the reaction diagram
B3V4Z0, -
-
-
-
?
Dnp-GGIR + H2O
?
show the reaction diagram
B3V4Z0, -
-
-
-
?
Dnp-GGK + H2O
?
show the reaction diagram
B3V4Z0, -
-
-
-
?
Dnp-GGLR + H2O
?
show the reaction diagram
B3V4Z0, -
-
-
-
?
fluorescein isothyocyanate-conjugated bovine serum albumin + H2O
?
show the reaction diagram
-
-
-
-
?
Furylacryloyl-Gly-Ile-NH2 + H2O
?
show the reaction diagram
-
-
-
-
-
furylacryloyl-Gly-Leu methyl ester + H2O
?
show the reaction diagram
-
-
-
-
-
furylacryloyl-Gly-Leu-NH2 + H2O
?
show the reaction diagram
-
-
-
-
-
furylacryloyl-Gly-Leu-NH2 + H2O
?
show the reaction diagram
-
-
-
-
-
Furylacryloyl-Leu-Leu-NH2 + H2O
?
show the reaction diagram
-
-
-
-
-
Furylacryloyl-Phe-Phe-NH2 + H2O
?
show the reaction diagram
-
-
-
-
-
Furylacryloyl-Thr-Leu-NH2 + H2O
?
show the reaction diagram
-
-
-
-
-
Gelatin + H2O
?
show the reaction diagram
-
-
-
-
?
Gelatin + H2O
?
show the reaction diagram
-
9% of the activity with denatured casein
-
-
?
Gelatin + H2O
?
show the reaction diagram
Bacillus megaterium A9542
-
-
-
-
?
Gly-Leu-NH2 + H2O
?
show the reaction diagram
-
-
-
-
-
N-(3-[2-furyl]acryloyl)-Gly-Leu amide + H2O
?
show the reaction diagram
-
-
-
?
nematode cuticle + H2O
?
show the reaction diagram
-
2.8% of the activity with denatured casein
-
-
?
Oxidized insulin B-chain + H2O
?
show the reaction diagram
-
major cleavages at the peptide bonds of His5-Leu6, His10-Leu11, Ala14-Leu15, Tyr16-Leu17, Gly23-Phe24 and Phe24-Phe25
-
-
-
plasminogen + H2O
angiostatin + mini-plasminogen
show the reaction diagram
-
cleavage mainly at S441-V442
-
-
?
protein C + H2O
?
show the reaction diagram
Bacillus megaterium, Bacillus megaterium A9542
-
-
-
-
?
prothrombin + H2O
thrombin + thrombin propeptide
show the reaction diagram
-
-
-
-
?
prourokinase + H2O
urokinase + urokinase propeptide
show the reaction diagram
-
-
-
-
?
skimmed milk + H2O
?
show the reaction diagram
-
60% of the activity with denatured casein
-
-
?
insulin B chain + H2O
?
show the reaction diagram
-
-
-
?
additional information
?
-
-
specificity overview: various synthetic peptides
-
-
-
additional information
?
-
-
specificity overview: peptide bonds in which leucine is involved by its amino group are rapidly split, then those of phenylalanine and lastly those of other hydrophobic residues. The residue involved in the bond by the carboxyl group may have a stimulating effect, not: benzoyl-Gly-Phe, benzoyl-Gly-Leu (carboxypeptidase A substrates), benzoyl-Gly-Arg, benzoyl-Gly-Lys (carboxypeptidase B substrates), amides (Gly-NH2, Ser-NH2, His-NH2, Arg-NH2, Met-NH2, Leu-NH2, Phe-NH2, Tyr-NH2, Trp-NH2), benzoyl-Arg ethyl ester, tosyl-Arg methyl ester, benzoyl-Tyr ethyl ester
-
-
-
additional information
?
-
B3V4Z0, -
analysis of substrate specificity of MprBi using synthetic chromogenic substrates, MALDI-TOF mass spectrometric product analysis, overview
-
-
-
additional information
?
-
-
C histolyticum neutral protease, CHNP, shows similar preferences for specific amino acid sequences as thermolysin, EC 3.4.24.27, and Bacillus polymyxa protease, which cleave on the amino terminal side of hydrophobic amino acids such as leucine
-
-
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Ca2+
-
100 mM, 60% activation
Ca2+
-
5 mM Ca2+ stabilizes the enzyme
Ca2+
B3V4Z0, -
30% activation at 10 mM
Calcium
-
2 calcium binding sites
Mg2+
B3V4Z0, -
20% activation at 10 mM
Zinc
-
zinc enzyme
Zinc
-
zinc enzyme
Zinc
-
zinc enzyme
Zn2+
-
Zn2+ ions, although essential cofactors and exhibiting extremely high affinity, are without effect on the stability
Zn2+
B3V4Z0, -
secreted zinc-dependent endopeptidase, addition of exogenous Zn2+ at 0.01 mM inhibits the enzyme
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
1,10-phenanthroline
B3V4Z0, -
94.2% inhibition at 0.5 mM, 100% at 5 mM
dithiothreitol
-
5 mM, 83% residual activity
EDTA
-
1 mM, complete inhibition
EDTA
B3V4Z0, -
4% inhibition at 0.5 mM, 94.3% at 5 mM
guanidine hydrochloride
-
competitive inhibitor
N-[alpha-L-rhamnopyranosyl-oxyhydroxyphosphinyl]-L-leucyl-L-tryptophan
-
-
Organic solvents
-
and related compounds, overview
-
PCMB
B3V4Z0, -
6% inhibition at 0.5 mM, 98.1% at 5 mM
phenylmethylsulfonyl fluoride
-
0.1 mM, 77% residual activity
PMSF
B3V4Z0, -
6% inhibition at 0.5 mM, 9% at 5 mM
Zn2+
-
1 mM, 80% loss of activity
Zn2+
B3V4Z0, -
secreted zinc-dependent endopeptidase, addition of exogenous Zn2+ at 0.01 mM inhibits the enzyme
HgCl2
B3V4Z0, -
49% inhibition at 0.5 mM, 60% at 5 mM
additional information
-
not inhibitory: 1 mM iodoacetic acid, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM leupeptin, 0.5 mM pepstatin, 0.005 mM alpah2-plasmin inhibitor
-
additional information
B3V4Z0, -
no inhibition by protein trypsin inhibitor
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
CoCl2
-
0.3 mM, 1.8fold increase in activity
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
1.3
-
3-(2-furylacryloyl)-glycyl-L-leucine amide
-
pH 7.5, 37C, wild-type
1.4
-
3-(2-furylacryloyl)-glycyl-L-leucine amide
-
pH 7.5, 37C, mutant I157D
0.25
-
3-(2-furylacryloyl)-L-alanyl-L-phenylalanine amide
-
pH 7.5, 37C, mutant I157D
0.3
-
3-(2-furylacryloyl)-L-alanyl-L-phenylalanine amide
-
pH 7.5, 37C, wild-type
0.06
-
azocasein
B3V4Z0, -
recombinant wild-type enzyme, pH 8.0, 37C
-
0.003
-
Plasminogen
-
pH 7.4, 25C
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
80
-
3-(2-furylacryloyl)-glycyl-L-leucine amide
-
pH 7.5, 37C, mutant I157D
256
-
3-(2-furylacryloyl)-glycyl-L-leucine amide
-
pH 7.5, 37C, wild-type
64
-
3-(2-furylacryloyl)-L-alanyl-L-phenylalanine amide
-
pH 7.5, 37C, wild-type
99
-
3-(2-furylacryloyl)-L-alanyl-L-phenylalanine amide
-
pH 7.5, 37C, mutant I157D
1210
-
azocasein
B3V4Z0, -
recombinant wild-type enzyme, pH 8.0, 37C
-
0.7
-
Plasminogen
-
pH 7.4, 25C
-
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
1000
-
guanidine hydrochloride
-
pH 7.5, 25C
0.0035
-
N-[alpha-L-rhamnopyranosyl-oxyhydroxyphosphinyl]-L-leucyl-L-tryptophan
-
pH 7.5, 37C, wild-type
0.0037
-
N-[alpha-L-rhamnopyranosyl-oxyhydroxyphosphinyl]-L-leucyl-L-tryptophan
-
pH 7.5, 37C, mutant I157D
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.0057
-
B3V4Z0, -
with substrate Dnp-AAVR, pH 8.0, 37C
0.0145
-
B3V4Z0, -
with substrate Dnp-GGLR, pH 8.0, 37C
0.0264
-
B3V4Z0, -
with substrate Dnp-GGK, pH 8.0, 37C
0.0282
-
B3V4Z0, -
with substrate Dnp-GGIR, pH 8.0, 37C
0.0365
-
B3V4Z0, -
with substrate Dnp-GGFR, pH 8.0, 37C
0.0515
-
B3V4Z0, -
with substrate Dnp-AALR-NH2, pH 8.0, 37C
17.4
-
B3V4Z0, -
with substrate azocasein, recombinant purified wild-type enzyme, pH 8.0, 37C
55.6
-
-
pH 6.5, 55C
78.5
-
-
Boilysin at 37C with casein as substrate
81.5
-
-
pseudo wild type at 37C with casein as substrate
81.9
-
-
wild type enzyme at 37C with casein as substrate
82.4
-
-
double mutant G8C/N60C at 37C with casein as substrate
242
-
-
wild type enzyme at temperature optimum 75C
273
-
-
pseudo wild type at temperature optimum 75C
375
-
-
double mutant G8C/N60C at temperature optimum 80C
608
-
-
boilysin at temperature optimum 95C
additional information
-
-
-
additional information
-
-
-
additional information
-
P43133
16530 microg Tyr produced per min, pH 7.5, 65C
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
8
-
B3V4Z0, -
asay at
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.5
-
P43133
sharp decrease in activity below
6.5
11
-
decrease of activity from pH 6.5 to 11
11
-
-
no residual activity
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
37
-
B3V4Z0, -
assay at
60
-
-
activity toward N-(3-[2-furyl]acryloyl)-Gly-Leu amide is 4 times higher than at 25C
75
-
-
pseudo wild type; wild type enzyme
80
-
-
double mutant G8C/N60C
TEMPERATURE RANGE
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
22
70
B3V4Z0, -
activity range
pI VALUE
pI VALUE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.4
-
B3V4Z0, -
-
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
B3V4Z0, -
secreted zinc-dependent endopeptidase
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
34500
-
-
Bacillus stearothermophilus NCIB 8924
35000
-
-
Bacillus stearothermophilus NRRL B-3880
additional information
-
-
primary structure of Bacillus subtilis and Bacillus cereus enzyme
additional information
-
-
of Bacillus mesentericus enzyme
additional information
-
-
active site structure predicted by computer-aided modeling on the base of the three-dimensional structure of thermolysin
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 40000, SDS-PAGE
?
-
x * 35300, SDS-PAGE
?
P43133
x * 35000, SDS-PAGE
?
B3V4Z0, -
x * 19000, SDS-PAGE
?
Bacillus megaterium A9542
-
x * 35300, SDS-PAGE
-
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
crystal structure at 3.0 A resolution
-
pH STABILITY
pH STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
4
-
-
1h, less than 40% remaining activity
4
-
-
60 min, complete loss of activity
5
8
-
stable within this range
5
9
-
37C, 60 min, stable
11.7
-
-
60 min, complete loss of activity
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
60
-
stable
30
60
-
stable for 30 min
58.6
-
-
T50 value at pH 7.0
59.5
-
-
T50 value at pH 5.3
60
-
-
15 min, 50% loss of activity without stabilizer, completely stable for 1 h in presence of 0.05 M CaCl2
65
-
-
Bacillus stearothermophilus NCIB 8924 enzyme stable at
65
-
P43133
1 h, 80% residual activity
70
-
-
Bacillus stearothermophilus B-3880 enzyme stable at
70
-
-
1 h, complete inactivation
70
-
-
20 min, 65% loss of activity
92.5
-
-
G8C/N60C double mutation extremely thermostable, half-life 35.9 min
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
Ca2+ stabilizes against thermal inactivation
-
MgCl2 stabilizes
-
native wild type and G8C/N60C enzymes are stable toward autoproteolysis and keep their structural integrity for several weeks
-
ORGANIC SOLVENT
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
2-propanol
-
48 h, 37C, 50% residual activity with 44.5% (v/v) and 65.5% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
Acetone
-
48 h, 37C, 50% residual activity with 30.0% (v/v) and 50.1% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
acetonitrile
-
48 h, 37C, 50% residual activity with 30.6% (v/v) and 51.6% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
dimethylformamide
-
48 h, 37C, 50% residual activity with 29.8% (v/v) and 45.8% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
dioxane
-
48 h, 37C, 50% residual activity with 32.1% (v/v) and 47.9% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
DMSO
-
48 h, 37C, 50% residual activity with 32.9% (v/v) and 48.7% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
Methanol
-
48 h, 37C, 50% residual activity with 56.6% (v/v) and 86.4% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant enzyme from Bacillus subtilis culture medium by ammonium sulfate fractionation and hydrophobic interaction chromatography to homogeneity
B3V4Z0, -
wild-type and mutant TLPs
-
extracellular enzyme from culture medium by several chromatographic steps including one affinity based step
-
after expression in Escherichia coli with N-terminal His-tag
-
recombinant enzyme
P43133
wild-type and mutant enzymes
-
extracellular enzyme from culture medium by anion exchange chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
gene contains a large open reading frame between the regions coding for signal sequence and mature protein
-
gene mprBi, DNA and amino acid sequence determination and analysis, enzyme expression in Bacillus subtilis and secretion to the culture medium
B3V4Z0, -
protease-deficient strain Bacillus subtilis DB117 used as host for plasmids, subcloned in Escherichia coli for site-directed mutagenesis
-
expression in Bacillus subtilis
P43133
nucleotide sequence and promoter region
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
I157D
-
site-directed mutagenesis
G8C/N60C
-
double mutation
G8C/N60C
-
introduction of disulfide bridge fixing the region near calcium binding site III. At guanidinium/HCl concentrations above 7.5 M, mutant unfolds distinctly more slowly than wild-type. Below 7.5 M guanidinium/HCl, mutant behaves similar to wild-type
G8C/N60C
-
double mutant temperature optimum at 80C, increased thermal stability
G8C/N60C
-
thermostable variant of the non-specific neutral protease
W55F
-
higher Ca2+ concentration is needed than for wild-type for occupation of Ca2+-binding site III at amino acids 55-59
Renatured/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
protease activity can be significantly regained if the completely unfolded enzyme is transferred from 8 M guanidine-HCl into native-like conditions (0.4 M guanidine-HCl) by dilution with buffer
-
renaturation of enzyme from inclusion bodies after solubilization using guanidine hydrochloride
-
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
synthesis
-
production of enzyme as an active mature enzyme in Escherichia coli in the absence of its prosequence. Expression of enzyme with N-terminal His-tag, which accumulates as inclusion bodies and renaturation after solubilization using guanidine hydrochloride