Information on EC 2.3.1.164 - isopenicillin-N N-acyltransferase

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The expected taxonomic range for this enzyme is: Trichocomaceae

EC NUMBER
COMMENTARY
2.3.1.164
-
RECOMMENDED NAME
GeneOntology No.
isopenicillin-N N-acyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
phenylacetyl-CoA + isopenicillin N + H2O = CoA + penicillin G + L-2-aminohexanedioate
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Acylation
-
-
-
-
hydrolysis
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
Metabolic pathways
-
Penicillin and cephalosporin biosynthesis
-
penicillin K biosynthesis
-
SYSTEMATIC NAME
IUBMB Comments
acyl-CoA:isopenicillin N N-acyltransferase
Proceeds by a two stage mechanism via 6-aminopenicillanic acid. Different from EC 3.5.1.11, penicillin amidase.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6-aminopenicillanate acyltransferase
-
-
-
-
6-aminopenicillanic acid acyltransferase
-
-
-
-
6-aminopenicillinanic acid phenylacetyltransferase
-
-
-
-
6-aminopenicillinanic acylase
-
-
-
-
aatA
-
gene encoding isopenicillin N acyltransferase
aatB
-
AN6775.3, gene encoding a penicillin V producing activity, which, in contrast to aatA, does not encode a peroxisomal targeting signal PTS1. aatB homologues also exist in non-penicillin-producing fungi and is suggested to be a paralogue of aatA.
acyl coA:isopenicillin N-acyltransferase
P15802
-
acyl coenzyme A: isopenicillin N acyltransferase
-
-
acyl coenzyme A:6-aminopenicillanic acid acyltransferase
-
-
-
-
acyl-CoA:6-aminopenicillanate acyltransferase
-
-
-
-
acyl-CoA:6-APA acyltransferase
-
-
-
-
acyl-CoA:isopenicillin N acyltransferase
-
-
-
-
acyl-coenzyme A: IPN acyltransferase
-
-
acyl-coenzyme A:isopenicillin N acyltransferase
-
-
-
-
acyl-coenzyme A:isopenicillin N-acyltransferase
-
-
-
-
acyltransferase, 6-aminopenicillanate
-
-
-
-
IAT
Penicillium chrysogenum DS17690
-
-
-
isopenicillin N acyltransferase
-
-
-
-
isopenicillin N acyltransferase
-
-
isopenicillin N acyltransferase
-
-
isopenicillin N acyltransferase
Penicillium chrysogenum DS17690
-
-
-
isopenicillin N:acyl CoA acyltransferase
-
-
isopenicillin N:acyl-CoA: acyltransferase
-
-
-
-
isopenicillin-N N-acyltransferase
-
-
CAS REGISTRY NUMBER
COMMENTARY
54576-90-8
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
C103A mutant enzyme which has lost its ability to be processed to the mature heterodimeric protein and thus represents the proenzyme form
-
-
Manually annotated by BRENDA team
expression in Hansenula polymorpha
-
-
Manually annotated by BRENDA team
high yielding strain
-
-
Manually annotated by BRENDA team
penDE genes; SC6140, ATCC 2044, source of native enzyme
-
-
Manually annotated by BRENDA team
Penicillium chrysogenum npe6 lacks isopenicillin N acyltransferase activity
-
-
Manually annotated by BRENDA team
Penicillium chrysogenum DS17690
gene penDE
-
-
Manually annotated by BRENDA team
Penicillium chrysogenum WIS 54-1255
Wis 54-1255
-
-
Manually annotated by BRENDA team
Penicillium chrysogenum yielding
high yielding strain
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
malfunction
-
penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid accumulates
malfunction
Penicillium chrysogenum DS17690
-
penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid accumulates
-
metabolism
-
catalyses final step of penicillin biosynthesis
physiological function
-
IAT catalyzes a limiting step in high-yielding penicillin production strains
physiological function
Penicillium chrysogenum DS17690
-
IAT catalyzes a limiting step in high-yielding penicillin production strains
-
metabolism
P15802, -
in the last step of penicillin biosynthesis, acyl-CoA:isopenicillin N acyltransferase catalyzes the conversion of isopenicillin N to penicillin G
additional information
P15802, -
substrate binding structure determined by homology modeling and molecular docking in different snapshots, and refined by molecular dynamic simulations. A site located in the region generated by beta1, beta2 and beta5 strands, which forms part of the central structure of beta-subunit, as the potential binding site of isopenicillin N, which establishes interactions with Cys103, Leu169, Gln172, Asp264, and Arg310, detailed overview
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
phenoxyacetyl-CoA + isopenicillin N + H2O
CoA + penicillin V + L-2-aminohexanedioate
show the reaction diagram
Penicillium chrysogenum, Penicillium chrysogenum DS17690
-
-
-
-
?
phenylacetyl-CoA + 6-aminopenicillanic acid + H2O
CoA + benzylpenicillin
show the reaction diagram
-
-
-
?
phenylacetyl-CoA + 6-aminopenicillanic acid + H2O
CoA + benzylpenicillin
show the reaction diagram
-
acyl-CoA:6-aminopenicillanate acyltransferase activity
-
?
phenylacetyl-CoA + 6-aminopenicillanic acid + H2O
CoA + benzylpenicillin
show the reaction diagram
Penicillium chrysogenum WIS 54-1255
-
-
-
?
phenylacetyl-CoA + isopenicillin N + H2O
CoA + benzylpenicillin + L-2-aminohexanedioate
show the reaction diagram
-
-, acyl-coenzyme A:isopenicillin N acyltransferase activity, S309 is involved in substrate acylation
-
?
phenylacetyl-CoA + isopenicillin N + H2O
CoA + penicillin G + L-2-aminohexanedioate
show the reaction diagram
-
-
-
-
?
phenylacetyl-CoA + isopenicillin N + H2O
CoA + penicillin G + L-2-aminohexanedioate
show the reaction diagram
P15802, -
-
-
-
?
phenylacetyl-CoA + isopenicillin N + H2O
CoA + penicillin G + L-2-aminohexanedioate
show the reaction diagram
P15802, -
IAT substitutes the alpha-aminoadipic acid side chain of isopenicillin N by a phenylacetic acid phenolate group from phenylacetyl-CoA
-
-
?
phenylacetyl-CoA + isopenicillin N + H2O
CoA + penicillin G + L-2-aminohexanedioate
show the reaction diagram
Penicillium chrysogenum DS17690
-
-
-
-
?
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
phenylacetyl-CoA + isopenicillin N + H2O
CoA + benzylpenicillin + L-2-aminohexanedioate
show the reaction diagram
-
-
-
?
phenylacetyl-CoA + isopenicillin N + H2O
CoA + penicillin G + L-2-aminohexanedioate
show the reaction diagram
-
-
-
-
?
phenylacetyl-CoA + isopenicillin N + H2O
CoA + penicillin G + L-2-aminohexanedioate
show the reaction diagram
P15802, -
-
-
-
?
phenylacetyl-CoA + isopenicillin N + H2O
CoA + penicillin G + L-2-aminohexanedioate
show the reaction diagram
Penicillium chrysogenum DS17690
-
-
-
-
?
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
-
-
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
8
-
-
activity assay
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
26
-
-
activity assay
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
additional information
-
the protein level of IAT already saturates at low cluster copy numbers, lack of correlation between the protein and transcript levels of the IAT enzyme
Manually annotated by BRENDA team
additional information
Penicillium chrysogenum DS17690
-
the protein level of IAT already saturates at low cluster copy numbers, lack of correlation between the protein and transcript levels of the IAT enzyme
-
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
IAT encoded by the aatB gene, lacking a peroxisomal targeting signal PTS1
Manually annotated by BRENDA team
-
colocalization of isopenicillin N-acyltransferase and phenylacetyl-CoA ligase
Manually annotated by BRENDA team
-
both native and recombinant enzyme
Manually annotated by BRENDA team
-
IAT encoded by the aatA gene, containing a peroxisomal targeting signal PTS1
Manually annotated by BRENDA team
Penicillium chrysogenum AS-P-78
-
colocalization of isopenicillin N-acyltransferase and phenylacetyl-CoA ligase
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
11000
-
-
processed protein, subunit alpha, determined by SDS-PAGE and Western blot analysis
28000
-
-
SDS-PAGE
29000
-
-
processed protein, subunit beta, determined by SDS-PAGE and Western blot analysis
40000
-
-
unprocessed precursor polypeptide, proenzyme, SDS-PAGE
40000
-
-
recominant enzyme SDS-PAGE and Western blot analysis
40000
-
-
isopenicillin N acyltransferase precursor, proacyltransferase, proIAT, determined by SDS-PAGE and Western blot analysis
40000
-
P15802, -
pro-enzyme
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
recombinant expression in Hansenula polymorpha give a 40 kDa pre-protein that is processed into an 11 and 29 kDa subunit, SDS-PAGE
dimer
-
alphabeta, 1 * 29000 + 1 * 11000, SDS-PAGE
dimer
-
1 * 11000 + 1 * 29000, SDS-PAGE
dimer
P15802, -
three-dimensional structure determined by homology modeling and molecular docking in different snapshots, and refined by molecular dynamic simulations, detailed overview. The gene for IAT, penDE, encodes a 357 residue proenzyme with a molecular weight of 40 kDa, which is processed autocatalytically to generate two polypeptides of 102 residues as alpha-subunit and 255 residues as beta-subunit
heterodimer
-
alpha 1 * 11000 + beta 1 * 29000, derived from cleavage of the 40 kDa polypeptide, SDS-PAGE
heterodimer
-
beta 1 * 29000, alpha subunit not detected
heterodimer
-
alpha 1 * 11000 + beta 1 * 29000, wild-type and recombinant enzyme, SDS-PAGE
heterodimer
Penicillium chrysogenum WIS 54-1255
-
beta 1 * 29000, alpha subunit not detected
-
additional information
-
isopenicillin N-acyltransferase and phenylacetyl-CoA ligase which supplies phenylacetic acid to the N-acyltransferase form a peroxisomal functional complex
additional information
Penicillium chrysogenum AS-P-78
-
isopenicillin N-acyltransferase and phenylacetyl-CoA ligase which supplies phenylacetic acid to the N-acyltransferase form a peroxisomal functional complex
-
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
proteolytic modification
-
the intronless gene is expressed in Escherichia coli and self-processed efficiently into its alpha and beta subunits
proteolytic modification
-
recombinant expression in Hansenula polymorpha give a 40 kDa pre-protein that is processed into an 11 and 29 kDa subunit
proteolytic modification
P15802, -
the proenzyme is processed autocatalytically to generate two polypeptides forming alpha-subunit and beta-subunit
additional information
-
ptoIAT overexpressed in Escherichia coli is not self-processed even when treated with Aspergillus nidulans crude extract
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
hanging-drop vapour-diffusion method, X-ray diffraction, monoclinic space group C2, unit-cell parameteres: a = 231.36 A, b = 68.27 A, c = 151.31 A, beta = 129.6° at -173°C
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
cell extracts of Penicillium chrysogenum are prepared, Escherichia coli soluble extracts are used for in vitro IAT activity measurement
-
recombinant wild-type enzyme with a C-terminal His-tag is purified in a single step using Ni2+ affinity chelate chromatography. A combination of 5 mM DTT + 250 mM NaCl + 5 mM EDTA is effective in preventing aggregation of the enzyme
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
overexpression in Escherichia coli, not self-processed even when treated with Aspergillus nidulans crude extract
-
expressed in Escherichia coli JM109
-
expression in Escherichia coli
-
gene penDE
P15802, -
gene penDE, lack of correlation between the protein and transcript levels of the IAT enzyme, quantitative real-time PCR expression analysis and gene copy number determination, overexpression of penDE in Penicillium chrysogenum strains DS47274, DS47273, and DS17690, introduction of penDE gene without introns into these strains in a random manner, via the nonhomologous end-joining pathway. Cooverexpression of penDE with gene phl, encoding the phenylacetic acid CoA ligase leading to production of phenoxyacetyl-CoA and penicillin V
-
pPenIAT and pPenIATC103S are used to express the penDE gene in Penicillium chrysogenum, pPBCalphabeta, pDEC103S and palphabeta-DEC103S are used for expression in Escherichia coli
-
the intronless gene is expressed in Escherichia coli and self-processed efficiently into its alpha and beta subunits
-
EXPRESSION
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
transcription factor AnBH1 acting as a repressor of aatA and aatB expression
-
transcription factor AnCF acting as an activator of aatA and aatB expression
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
C103S
-
unprocessed inactive variant of isopenicillin N acyltransferase
S227A
-
produces uncleaved proenzyme lacking enzyme activity
S309A
-
recombinant proenzyme containing S309A is cleaved, however enzyme activity is not observed
additional information
-
disruption of the aatA gene reduces penicillin production to 20%, the remaining activity is attributed to the product of gene aatB; Overexpression of a mutated aatB-PTS1 gene in an aatA-disruption strain, leading to peroxisomal localization of AatB, increased the penicillin titre more than overexpression of the wild-type aatB.
C103S or A or W
-
C103 is specifically required for recombinant proenzyme cleavage
additional information
-
in high-yielding penicillin production strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme. Cooverexpression of penDE with gene phl, encoding the phenylacetic acid CoA ligase leading to production of phenoxyacetyl-CoA and penicillin V
T105V
-
results in a significant inhibition of proenzyme cleavage
additional information
Penicillium chrysogenum DS17690
-
in high-yielding penicillin production strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme. Cooverexpression of penDE with gene phl, encoding the phenylacetic acid CoA ligase leading to production of phenoxyacetyl-CoA and penicillin V
-
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
biotechnology
-
Penicillium chrysogenum npe6 lacking isopenicillin N acyltransferase activity is an excellent host for production of different beta-lactam antibiotics