Information on EC 1.21.3.1 - isopenicillin-N synthase

New: Word Map on EC 1.21.3.1
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Mark a special word or phrase in this record:
Search Reference ID:
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)


The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
1.21.3.1
-
RECOMMENDED NAME
GeneOntology No.
isopenicillin-N synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine + O2 = isopenicillin N + 2 H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cyclization
-
-
oxidation
oxidative cyclization
redox reaction
-
-
-
-
reduction
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
-
-
isopenicillin N biosynthesis
-
-
Metabolic pathways
-
-
Penicillin and cephalosporin biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine:oxygen oxidoreductase (cyclizing)
Forms part of the penicillin biosynthesis pathway (for pathway, click here).
CAS REGISTRY NUMBER
COMMENTARY hide
78642-31-6
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain C10, ATCC 48278
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
i.e. Nocardia lactamdurans, NRRL 3802 var. JC 1843, cephamycin producing strain, Amy+ mutant
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
strain SC 12.154
-
-
Manually annotated by BRENDA team
strain SC 12.154
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
strain NRRL 3584
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
adipyl-L-cysteinyl-D-valine + ?
N-(4-carboxybutyl)penicillin + 2 H2O
show the reaction diagram
-
-
-
-
?
delta-(L-alpha-aminoadipoyl)-(3R)-methyl-L-cysteine D-alpha hydroxyvaleryl ester + O2
? + H2O
show the reaction diagram
-
-
-
-
?
delta-(L-alpha-aminoadipoyl)-L-cysteinyl-beta-methyl-D-cyclopropylglycine + O2
(2R,8R)-8-([(5S)-5-amino-5-carboxypentanoyl]amino)-3-methylene-9-oxo-6-thia-1-azabicyclo[5.2.0]nonane-2-carboxylic acid + H2O
show the reaction diagram
-
-
-
-
?
delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-hydroxyisovaleryl ester + O2
?
show the reaction diagram
-
for this substrate analogue (ACOV) lacking an amide nitrogen IPNS exhibits oxygenase activity
-
-
?
delta-(L-alpha-aminoadipoyl)-L-cysteinyl-O-methyl-D-allo-threonine + O2
(2S,3S,5R,6R)-6-[[(5S)-5-amino-5-carboxypentanoyl]amino]-3-methoxy-3-methyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid + 2 H2O
show the reaction diagram
delta-(L-alpha-aminoadipoyl)-L-homocysteinyl-D-cysteine + O2
(4S,7S)-7-[[(5S)-5-amino-5-carboxypentanoyl]amino]-6-oxohexahydropyrrolo[2,1-c][1,2,4]dithiazine-4-carboxylic acid + 2 H2O
show the reaction diagram
delta-(L-alpha-aminoadipoyl)-L-homocysteinyl-D-valine + O2
(2S)-2-amino-6-([(3S)-1-[(1R)-1-carboxy-2-methylpropyl]-5-hydroxy-2-oxopyrrolidin-3-yl]mino)-6-oxohexanoic acid + ?
show the reaction diagram
delta-(L-alpha-aminoadipoyl)-L-homocysteinyl-delta-S-methylcysteine + O2
?
show the reaction diagram
delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-alpha-aminobutyrate + O2
?
show the reaction diagram
-
wild-type and mutants, reaction mechanism
-
-
ir
delta-L-alpha-aminoadipoyl-L-cysteine (1-(S)-carboxy-2-thiomethyl)ethyl ester + O2
? + H2O
show the reaction diagram
-
substrate analogue
-
-
?
delta-L-alpha-aminoadipoyl-L-cysteinyl-D-valine + O2
?
show the reaction diagram
-
-
-
?
delta-L-alpha-aminoadipoyl-L-cysteinyl-D-valine + O2
isopenicillin N + 2 H2O
show the reaction diagram
L-alpha-aminoadipoyl-L-cysteinyl-D-valine + O2
? + H2O
show the reaction diagram
-
-
-
-
?
L-alpha-aminoadipoyl-L-cysteinyl-D-valine + O2
isopenicillin N + 2 H2O
show the reaction diagram
-
-
-
-
?
N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine + O2
isopenicillin + H2O
show the reaction diagram
N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine + O2
isopenicillin N + 2 H2O
show the reaction diagram
N6-[(1R,2S)-1-([[(1R)-1-carboxy-2-methylpropyl]oxy]carbonyl)-2-sulfanylpropyl]-6-oxo-L-lysine + O2
? + H2O
show the reaction diagram
-
substrate analogue
-
-
?
phenylacetyl-L-cysteinyl-D-valine + O2
penicillin G + 2 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
adipyl-L-cysteinyl-D-valine + ?
N-(4-carboxybutyl)penicillin + 2 H2O
show the reaction diagram
-
-
-
-
?
delta-L-alpha-aminoadipoyl-L-cysteinyl-D-valine + O2
isopenicillin N + 2 H2O
show the reaction diagram
-
-
-
-
?
L-alpha-aminoadipoyl-L-cysteinyl-D-valine + O2
? + H2O
show the reaction diagram
-
-
-
-
?
L-alpha-aminoadipoyl-L-cysteinyl-D-valine + O2
isopenicillin N + 2 H2O
show the reaction diagram
-
-
-
-
?
N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine + O2
isopenicillin + H2O
show the reaction diagram
-
-
-
-
?
N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine + O2
isopenicillin N + 2 H2O
show the reaction diagram
phenylacetyl-L-cysteinyl-D-valine + O2
penicillin G + 2 H2O
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
in Penicillium chrysogenum, the enzymes involved in penicillin production are compartmentalized in the cytosol and in microbodies
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
alpha-aminoadipic(-Cys-Gly)
bis[alpha-aminoadipic(-Cys-D-chloroalanine)]
bis[alpha-aminoadipic(-Cys-D-hexafluorovaline)]
bis[alpha-aminoadipic(-Cys-D-Phe)]
bis[alpha-aminoadipic(-Cys-D-Trp)]
bis[alpha-aminoadipic(-Cys-D-Tyr)]
bis[alpha-aminoadipic(-Cys-DL-hexafluorovaline)]
bis[alpha-aminoadipic(-Cys-hexafluorovaline)]
bis[H-Cys-D-Val]
Cu2+
-
slight inhibition
cystamine
-
100% inhibition at 0.5 mM
cysteamine
-
43% inhibition at 1.5 mM
cysteine
-
60% inhibition at 1 mM
cystine
-
81% inhibition at 1.5 mM
D-glucose 6-phosphate
-
strong inhibition
delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-cyclopropylglycine
-
-
delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-methionine
-
substrate analogue, which incorporates a thioether in place of the valinyl sidechain. Crystal structure of the enzyme in complex with the compound and Fe(II) at 1.40 A resolution reveals that the compound binds in the active site such that the sulfur atom of the methionine thioether binds to iron in the oxygen binding site at a distance of 2.57 A. The sulfur of the cysteinyl thiolate sits 2.36 A from the metal
delta-(L-alpha-aminoadipoyl)-L-cysteinyl-O-methyl-D-threonine
-
substrate analogue, not turned over by IPNS
glutathione
-
slight inhibition
GSH
-
70% inhibition at 1 mM
N-ethylmaleimide
N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine
-
substrate inhibition above 5 mM
NH4+
-
inhibition of enzyme formation in vivo, no inhibitory effect in vitro
Ni2+
-
moderate inhibition
pyruvate
-
slight inhibition
Triton X-100
-
inhibits at concentration of 0.5%
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ascorbate
-
reducing agent stimulates IPNS activity
ascorbic acid
dithioerythritol
-
absolutely required, highly stimulating
dithiothreitol
DTT
-
reducing agent stimulates IPNS activity
polyethylene glycol 1500
-
20% stimulation up to concentration of 5%
-
Triton X-100
-
leads to 50% stimulation at concentration of 0.01%
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.08 - 0.18
delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine
0.12 - 0.18
N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine
3.6
phenylacetyl-L-cysteinyl-D-valine
-
-
additional information
N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine
-
Km values for ACV exhibit for different cyclases are on the order of 0.2-0.3 mM
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.1
N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine
Aspergillus nidulans
-
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.9
N-ethylmaleimide
-
about
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0000000125
-
mutant C104S, recombinant, partially purified
0.000000093
-
purified recombinant enzyme
0.000000177
-
mutant C251S, recombinant, partially purified
0.000000257
-
mutant C142S, recombinant, partially purified
0.000000276
-
mutant C37S, recombinant, partially purified
0.000000338
-
wild-type, recombinant, partially purified
0.00000224
-
mutant H49L
0.0000027
-
wild-type
0.00000664
-
purified recombinant enzyme
0.0000103
-
mutant H126L
0.0000112
-
mutant H137L
0.0000116
-
mutant H116L
0.0000127
-
mutant H64L
0.0000136
-
wild-type
0.00129
-
about, purified enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9
-
30% activity remaining above pH 9.0, highly reduced activity below pH 6.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
26500
-
gel filtration
36500 - 38000
-
gel filtration and SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of the enzyme reveals that the active site of IPNS is buried in a characteristic jelly-roll motif that has been found in other oxygenases
-
crystal structure of the enzyme in complex with substrate analoge delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-methionine and Fe(II) at 1.40 A resolution reveals that the compound binds in the active site such that the sulfur atom of the methionine thioether binds to iron in the oxygen binding site at a distance of 2.57 A. The sulfur of the cysteinyl thiolate sits 2.36 A from the metal
-
enzyme complexed with manganese instead of iron in the active site, more stable; structure analysis
in complex with substrat analogue delta-(L-alpha-aminoadipoyl)-L-cysteinyl-O-methyl-D-threonine and Fe(II). Structure reveals an additional water molecule bound to the active site metal, held by hydrogen-bonding to the ether oxygen atom of the substrate analogue
-
in complex with substrate analogue delta-(L-alpha-aminoadipoyl)-(3R)-methyl-L-cysteine D-alpha hydroxyvaleryl ester, crystallization with anaerobic conditions and exposure of crystals to oxygen giving a hydroxymethyl/ene product. Discussion of steric and electronic effects around the valinyl isopropyl side chain of the enzymes active side
-
in complex with substrate homologues delta-(L-alpha-aminoadipoyl)-L-homocysteinyl-D-valine and delta-(L-alpha-aminoadipoyl)-L-homocysteinyl-delta-S-methylcysteine. The complex with Fe(II) and delta-(L-alpha-aminoadipoyl)-L-homocysteinyl-D-valine shows diffuse electron density for several regions of the substrate, revealing considerable conformational freedom within the active site. The substrate is more clearly resolved in the complex delta-(L-alpha-aminoadipoyl)-L-homocysteinyl-delta-S-methylcysteine by virtue of thioether coordination to iron. delta-(L-alpha-aminoadipoyl)-L-homocysteinyl-delta-S-methylcysteine occupies two distinct conformations, both distorted relative to the natural substrate (L-alha-aminoadipoyl)-L-cysteinyl-D-valine, in order to accommodate the extra methylene group in the second residue
-
in complex with tripeptyl analogues delta-(L-alpha-aminoadipoyl)-L-cysteinyl-beta-methyl-D-cyclopropylglycine and delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-cyclopropylglycine, crystallization in presence of Fe-(II) under anaerobic conditions
-
in complex with truncated substrate analogues delta-(L-alpha-aminoadipoyl)-L-cysteinyl-glycine and delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alanine in presence of Fe(II) and presence and absence of nitric oxide. C-terminal carboxylate of substrate is oriented toward the active site iron atom
-
structure analysis
-
the crystal structures of isopenicillin N synthase in complex with gamma-(L-alpha-aminoadipoyl)-(3S-methyl)-L-cysteine D-alpha-hydroxyisovaleryl ester and FeII exposed to O2 and unexposed to O2 are solved to resolutions of 2.2 and 1.65 A, respectively
-
the crystal structures of isopenicillin N synthase in complex with L-alpha-aminoadipoyl-L-cysteine (1-(S)-carboxy-2-thiomethyl)ethyl ester and FeII exposed to O2 and unexposed to O2 are determined to 1.4 and 1.8 A resolutions, respectively
-
crystal structure, molecular modeling of the active site structure and the Fe2+-binding motif
-
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
unstable to 20 mM dithiothreitol during storage at -20C, 10% activity remaining
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme is unstable to oxidizing oxygen species in the reaction solution
-
440327
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gel filtration
-
partially, wild-type and mutants recombinant from Escherichia coli
-
recombinant from Escherichia coli
-
recombinant wild-type and mutants from Escherichia coli
-
recombinant, solubilized enzyme
-
wild-type and mutants are purified as catalytically latent apoenzymes
-
wild-type and mutants recombinant from Escherichia coli
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
-
expression in Escherichia coli
expression of wild-type and mutant in Escherichia coli, evaluation of growth temperature for expression of the soluble mutant and wild-type in the Escherichia coli host BL21 (DE3)
-
expression of wild-type and mutants as maltose-binding fusion proteins in Escherichia coli
-
expression of wild-type and mutants in Escherichia coli
-
expression of wild-type and mutants in Escherichia coli, amino acid sequence comparison
-
functional expression in the cytosol of yeast Hansenula polymorpha, best at 25C or 30C growth temperature, at 37C the recombinant protein is not stable, optimization of culture conditions, overview
-
overexpression in Escherichia coli K12 strain JM109 in inclusion bodies
-
overexpression in Escherichia coli, DNA sequence analysis
-
overexpression of recombinant enzyme mutants in Cephalosporium acremonium
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
a developed gene regulation model predicts the expression of this rate limiting enzyme based on glucose repression, fast decay of the mRNA encoding for the enzyme as well as the decay of the enzyme itself
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C106S
-
site-directed mutagenesis, 63% reduced activity, 14fold increased Km for N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine
C106S/C255S
-
site-directed mutagenesis, 63% reduced activity, 14fold increased Km for N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine
C255S
-
site-directed mutagenesis, 33% reduced activity, 1.4fold increased Km for N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine
H116L
-
site-directed mutagenesis, reduced activity
H126L
-
site-directed mutagenesis, reduced activity
H137L
-
site-directed mutagenesis, reduced activity
H262L
-
site-directed mutagenesis, complete loss of activity
H272L
-
site-directed mutagenesis, complete loss of activity
H49L
-
site-directed mutagenesis, complete loss of activity
H64L
-
site-directed mutagenesis, reduced activity
P285L
-
site-directed mutagenesis, complete loss of activity, increased soluble expression in Escherichia coli host
Q227L
-
site-directed mutagenesis, 55.10% remaining activity compared to wild-type
Q337L
-
site-directed mutagenesis, slightly reduced activity compared to wild-type
Q230L
-
mutant of IPNS shows diminished enzyme activity, residue is highly conserved among dioxygenases and proximal to the active site, is the fourth ligand for the Fe2+ atom
D216E
-
mutant, retains 1% activity
L223A
-
reduced activity with N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate is a poor substrate
L223I
-
reduced activity with N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine, product spectrum differs from that of the wild-type with delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate as substrate
L223V
-
reduced activity with N-[(5S)-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine, product spectrum differs from that of the wild-type with delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate as substrate
C104S
-
single-strand-site-directed mutagenesis, loss of more than 96% activity
C142S
-
single-strand-site-directed mutagenesis, loss of 24% activity
C251S
-
single-strand-site-directed mutagenesis, loss of 47.7% activity
C37S
-
single-strand-site-directed mutagenesis, loss of 18.3% activity
C37S/C142S/C251S
-
triple mutant, conformationally different from wild-type, prepared by recombining fragments of IPNS-encoding gene pcbC from each of the single mutants, loss of more than 90% activity
D214C
-
site-directed mutagenesis, active site mutant, complete loss of activity
D214E
-
site-directed mutagenesis, active site mutant, retains 1% of activity compared to wild-type
H212D
-
site-directed mutagenesis, active site mutant, complete loss of activity
H212N
-
site-directed mutagenesis, active site mutant, complete loss of activity
H212Q
-
site-directed mutagenesis, active site mutant, complete loss of activity
H268D
-
site-directed mutagenesis, active site mutant, complete loss of activity
H268N
-
site-directed mutagenesis, active site mutant, complete loss of activity
H268Q
-
site-directed mutagenesis, active site mutant, complete loss of activity
additional information
-
exchange of Asp214 and His268, and exchange of Asp214 and His 212 by site-directed mutagenesis leads to completely inactive enzymes
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
dissolution of the recombinant enzyme from Escherichia coli inclusion bodies with 7 M urea containing 20 mM DTT, 30 mM glycine, pH 10.0, requires vigorous stirring for over 2 hours at room temperature
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
-
production of beta-lactam antibiotics
medicine
-
isopenicillin N synthase is a potential target in the design of novel antibiotic compounds
pharmacology
synthesis
-
direct enzymic synthesis of antibiotics