Báo cáo khoa học: The isopenicillin N acyltransferases of Aspergillus nidulans and Penicillium chrysogenum differ in their ability to maintain the 40-kDa ab heterodimer in an undissociated form pdf

The isopenicillin N acyltransferases of
Aspergillus nidulans
and
Penicillium chrysogenum
differ in their ability to maintain
the 40-kDa ab heterodimer in an undissociated form
Francisco J. Ferna
´
ndez
1
, Rosa E. Cardoza
2
, Eduardo Montenegro
1
, Javier Velasco
1
, Santiago Gutie
´
rrez
1,2
and Juan F. Martı
´
n
1,2
1
Area de Microbiologı
´
a, Facultad de Ciencias Biolo
´
gicas y Ambientales, Universidad de Leo
´

n, Spain;
2
Instituto de Biotecnologı
´
ade
Leo
´
n INBIOTEC, Parque Cientı
´
fico de Leo
´
n, Spain
The isopenicillin N acyltransferases (IATs) of Aspergillus
nidulans and Penicillium chrysogenum differed in their ability
to maintain the 40-kDa proacyltransferase ab heterodimer
in an undissociated form. The native A. nidulans IAT
exhibited a molecular mass of 40 kDa by gel filtration. The
P. chrysogenum IAT showed a molecular mass of 29 kDa by
gel filtration (corresponding to the b subunit of the
enzyme) but the undissociated 40-kDa heterodimer was
never observed even in crude extracts. Heterologous
expression experiments showed that the chromatographic
behaviour of IAT was determined by the source of the
penDE gene used in the expression experiments and not by
the host itself. When the penDE gene of A. nidulans was
expressed in P. chrysogenum npe6andnpe8orinAcremo-
nium chrysogenum, the IAT formed had a molecular mass of
40 kDa. On the other hand, when the penDE gene origin-
ating from P. chrysogenum was expressed in A. chryso-
genum, the active IAT had a molecular mass of 29 kDa. The

intronless form of the penDE gene cloned from an A. nidu-
lans cDNA library and overexpressed in Escherichia coli
formed the enzymatically active 40-kDa proIAT, which was
not self-processed as shown by immunoblotting with anti-
bodies to IAT. This 40-kDa protein remained unprocessed
even when treated with A. nidulans crude extract. In con-
trast, the P. chrysogenum penDE intronless gene cloned
fromacDNAlibrarywasexpressedinE. coli,andtheIAT
was self-processed efficiently into its a (29 kDa) and
b (11 kDa) subunits. It is concluded that P. chrysogenum
and A. nidulans differ in their ability to self-process their
respective proIAT protein and to maintain the a and
b subunits as an undissociated heterodimer, probably
because of the amino-acid sequence differences in the
proIAT which affect the autocatalytic activity.
Keywords: enzyme processing; filamentous fungi; gene
expression; penicillin biosynthesis.
Aspergillus nidulans and Penicillium chrysogenum are able
to synthesize hydrophobic penicillins because of substitu-
tion of the
L
-a-aminoadipyl side chain of isopenicillin N by
aromatic acyl side chains catalyzed by the isopenicillin N
acyltransferase (IAT) [1–3], whereas Acremonium chryso-
genum lacks this enzyme [4]. The genes encoding the three
enzymes of the penicillin biosynthetic pathway pcbAB [for
the multienzyme d(a-aminoadipyl)-cysteinyl-valine synthe-
tase], pcbC (for the isopenicillin N synthase) and penDE
(for IAT) have been cloned from P. chrysogenum [5–10]
and A. nidulans [11–13] and are linked in a cluster [8]. The

three genes were found to be very similar in A. nidulans
and P. chrysogenum, and the overall organization of the
penicillin gene cluster is identical in the two fungi [14,15].
However, wild-type strains of P. chrysogenum areableto
synthesize 30-fold higher levels of penicillin than wild-type
A. nidulans (their penicillin production levels are about 150
and 5 lgÆmL
)1
in shake flasks, respectively) implying that
differences in expression of the penicillin biosynthesis
genes, or changes in enzyme processing or enzyme activity,
may be responsible for the disparity in penicillin biosyn-
thetic ability.
The last enzyme of the penicillin biosynthetic pathway,
IAT, has been shown to be a complex protein which
catalyzes five related reactions [16]. The P. chrysogenum
IAT is a heterodimer [17,18] composed of two subunits,
a (11 kDa, corresponding to the N-terminal region) and
b (29 kDa, C-terminal region), which are formed from a
40-kDa precursor protein (proacyltransferase) encoded by
the penDE gene [6]. The enzymatic activities and substrate
specificity of the proacyltransferase compared with that of
the mature enzyme remain obscure. Initial studies on the
purification of IAT showed that activity is associated with
the 29-kDa subunit [19,20]. Later, Tobin and coworkers
[17,18] showed that a higher IAT activity is observed after
association of the 29-kDa and 11-kDa subunits, i.e. the
most active P. chrysogenum IAT is a heterodimer of these
two subunits.
Our initial experiments indicated that the chromato-

graphic behaviour of A. nidulans IAT differed from that of
P. chrysogenum. This prompted us to investigate whether
Correspondence to J. F. Martı
´
n, Area de Microbiologı
´
a,
Facultad de Ciencias Biolo
´
gicas y Ambientales, Universidad de Leo
´
n,
24071 Leo
´
n, Spain. Fax: + 34 987 291506,
E-mail:
Abbreviations: IAT, isopenicillin N acyltransferase; IPTG, isopropyl
thio-b-
D
-galactoside.
(Received 31 October 2002, revised 10 December 2002,
accepted 10 March 2003)
Eur. J. Biochem. 270, 1958–1968 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03561.x
there were differences in the post-translational processing of
the proIATs of the two fungi that might explain the
differences in their penicillin biosynthetic ability. For this
purpose, antibodies against the P. chrysogenum and
A. nidulans proacyltransferases were prepared.
Our study shows that the A. nidulans proIAT remains
undissociated as a 40-kDa protein through various purifi-

cation steps, whereas the P. chrysogenum enzyme is effi-
ciently self-processed, rapidly dissociating into the 29-kDa
and 11-kDa subunits. The IAT of A. nidulans purified from
Escherichia coli expressing the intronless form of the penDE
gene was fully active but was not self-processed. In contrast,
the P. chrysogenum IAT obtained in E. coli from an
intronless form of its gene was processed into its component
subunits.
Materials and methods
Strains
A. nidulans ATCC 28901, a biotin-requiring penicillin-
producing strain, was used for biochemical studies and as
a source of RNA for constructing the cDNA library. The
RNA was obtained from 72-h cultures in MFA medium
(containing in gÆL
)1
, pharmamedia 25, lactose 70, ammo-
nium sulfate 7.5, calcium carbonate 10, biotin 0.02 and
potassium phenoxyacetate 6.5, pH 7.2). Similarly, a cDNA
library of P. chrysogenum Wis49-408 was obtained from
total RNA of 48-h cultures in complex penicillin production
CP medium [21].
E. coli XL1-Blue [22], SURE and SOLR (Stratagene,
La Jolla, CA, USA) were used for construction of cDNA
libraries. E. coli JM109(DE3) [23], a strain that contains a
hybrid RNA polymerase gene (lacUV5 promoter coupled
to the T7 RNA polymerase gene) integrated into the
chromosome (Promega, Madison, WI, USA), was used
for isopropyl thiogalactoside-mediated induction of gene
expression from the pT7-7 vectors mediated by isopropyl

thio-b-
D
-galactoside (IPTG).
The phage kZAP II was used to clone cDNA inserts.
These inserts were recovered as either plasmids or phages.
The M13 and f1 phage derivatives ExAssist and VCSM13
(Stratagene)wereusedinthein vivo excision of the
recombinant cDNA clones.
Cell extracts
Cell extracts were obtained from 48-h or 72-h cultures of
A. nidulans ATCC 28901 or P. chrysogenum AS-P-78 (a
high-penicillin-producing strain donated by Antibio
´
ticos
S.A., Milan, Italy) in CP medium, which supports
efficient penicillin production. Cultures were incubated
at 25 °C in a rotary shaker at 250 r.p.m. as described
previously [12].
The mycelia were collected by filtration, washed three
times with sterile saline solution (9 gÆL
)1
NaCl)at4°C,
suspended (420 g wet weight) in TD buffer (50 m
M
Tris/
HClpH8.0,5m
M
dithiothreitol) and disrupted with glass
beads in a mechanical cell disruptor (Braun, Melsungen,
Germany) under liquid CO

2
refrigeration. The extract
was centrifuged at 20 000 g for 20 min, and the super-
natant collected and used for further purification.
Self-processing of
A. nidulans
IAT in cell extracts
In experiments to study IAT self-processing ability, extracts
of A. nidulans from 72-h cultures containing 7.92 mg
proteinÆmL
)1
were used. After incubation at different
temperatures, 50 lg protein was loaded into each well and
immunodetected after electrophoresis with a 1 : 80 000
dilution of antibodies to IAT.
IAT assays
The activity of A. nidulans or P. chrysogenum IAT was
quantified routinely as described previously [16]. One unit is
defined as the activity that forms 1 nmol penicillin GÆmin
)1
.
Specific activities are given as UÆ(mg protein)
)1
.
The IAT of either P. chrysogenum or A. nidulans was
purified after removal of nucleic acids by precipitation with
protamine sulfate in 50 m
M
Tris/HCl, pH 8.0 (final con-
centration 0.4%) followed by centrifugation at 20 000 g for

30 min. The proteins in the supernatant were fractionated
by precipitation with ammonium sulfate. Most IAT activity
was found in the 40–55% ammonium sulfate fraction.
Determination of molecular mass
ThemolecularmassoftheIATswasestablishedbygel
filtration of cell-free extracts on a Sephadex G75 Superfine
column (2.6 · 70 cm) calibrated with a set of molecular
mass markers (BSA, 67 kDa; ovalbumin, 43 kDa; chymo-
trypsinogen, 25 kDa, and ribonuclease A, 13.7 kDa).
SDS/PAGE
SDS/PAGE was performed as described by Laemmli [24].
Phosphorylase B (94 kDa), BSA (67 kDa), ovalbumin
(43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor
(20.1 kDa) and a-lactalbumin (14.4 kDa) (Pharmacia low
molecular mass calibration kit) were used as markers.
Expression in
E. coli
and refolding of IAT
Transformants of E. coli JM109(DE3) with the appropriate
cDNA constructions of the penDE genes of P. chrysogenum
or A. nidulans (see Results) were grown in Luria–Bertani
broth/chloramphenicol at 37 °CuntilanA
600
of 0.4 was
reached. Expression of the penDE gene was induced by the
addition of IPTG (final concentration 0.5 m
M
), and after
4 h the induced E. coli cells were harvested by centrifuga-
tion. Then 0.5 g E. coli cells were suspended in 10 mL

25 m
M
Tris buffer containing 5 m
M
EDTA,pH5.0,and
lysed by incubation with lysozyme (final concentration
1mgÆmL
)1
)for15minat4°C followed by sonication. The
insoluble inclusion bodies were collected by centrifugation
at 12 000 g for 10 min and solubilized in 8
M
urea in redox
buffer (containing 10 m
M
glutathione, 1 m
M
oxidised
glutathione, 50 m
M
Tris, 10 m
M
CaCl
2
and 10 m
M
MgCl
2
)
at pH 8.0.

Proteins were refolded from solubilized inclusion bodies
by diluting the redox buffer to a final concentration of 4
M
urea and 100 lgproteinÆmL
)1
, and supplementing it with
poly(ethylene glycol) 6000 [at a final molar ratio of
poly(ethylene glycol) to protein of 10] [25].
Ó FEBS 2003 Processing of the isopenicillin N acyltransferase (Eur. J. Biochem. 270) 1959
Preparation of antibodies against the
P. chrysogenum
or
A. nidulans
IATs
After expression of the corresponding genes in pT7-7
vectors [26] the purified inclusion bodies were solubilized
and the IAT was purified by semipreparative SDS/PAGE.
New Zealand rabbits were immunized by intradermal
injection with the pure protein, using the protocol described
by Dunbar & Schwoebel [27]. This immunization process
was repeated once a month for 3 months using incomplete
Freund’s adjuvant. After the immunization was completed,
blood serum was collected by centrifugation, and the IgG
fraction purified by ammonium sulphate precipitation and
FPLC using a Protein A–Sepharose column (Pharmacia
Biotech Inc., Uppsala, Sweden) as described by Harlow &
Lane [28].
Results
Molecular mass of the
A. nidulans

and
P. chrysogenum
IAT
Gel-filtration chomatography on Sephadex G75 superfine
of the A. nidulans IAT revealed a K
av
of 0.171, which
corresponded to a molecular mass of 39 811 Da, suggesting
that it is not dissociated into its component subunits. In
contrast, the P. chrysogenum IAT was eluted from the same
column with a K
av
of 0.275, which corresponded to a
molecular mass of 29 227 Da (Fig. 1A).
Processing and dissociation of the protein is determined
by the cloned gene and not by the host itself in A. nidulans
also. To establish if the lack of processing and dissociation
of the A. nidulans IAT was due to intrinsic characteristics
of the protein itself or to the absence of a processing
endopeptidase in the host, the penDE gene of either
P. chrysogenum or A. nidulans was introduced with its
own promoter and regulatory sequences into strains npe6
and npe8ofP. chrysogenum (deficient in IAT activity
[29,30]) and into A. chrysogenum CW19, a fungus that lacks
IAT [4].
Transformants with constructions containing either the
P. chrysogenum or A. nidulans penDE gene were selected,
and the molecular mass of the IAT was studied by
gel filtration through Sephadex G75. All transformants
Fig. 1. Comparative molecular mass of P. chrysogenum and A. nidulans

IATs when the corresponding genes are expressed in different fungal
hosts. (A) K
av
values deduced by gel filtration on Sephadex G75
superfine of the IATs of A. nidulans and P. chrysogenum,andseveral
transformants with the penDE gene. Strain designation AN28901
corresponds to A. nidulans 28901; W54-1255 is P. chrysogenum Wis54-
1255; CW19.3, 8p-1.3 and 6p-1.13 correspond to transformants of
A. chrysogenum CW19, P. chrysogenum npe8andP. chrysogenum
npe6, respectively, with the penDE gene of P. chrysogenum (molecular
mass 29 kDa). Strains CW19.10, 8B1 and 6A2 correspond, respect-
ively, to the IATs of A. chrysogenum CW19, P. chrysogenum npe8and
P. chrysogenum npe6 transformants with the penDE gene of A. nidu-
lans (molecular mass 40 kDa). Ribonuclease A (RA), chymotryp-
sinogen A (ChT), ovalbumin (OA), and BSA were used as standards.
All transformants were obtained with the corresponding native penDE
gene from P. chrysogenum or A. nidulans with their own promoters.
(B) Scheme summarizing the host strains and transformants used and
the molecular mass of their respective IATs. Dark boxes indicate
transformants with the penDE gene of A. nidulans and grey shading
corresponds to transformants with the P. chrysogenum penDE gene.
1960 F. J. Ferna
´
ndez et al.(Eur. J. Biochem. 270) Ó FEBS 2003
contained one to three integrated intact copies of either the
P. chrysogenum or A. nidulans penDE genes as shown by
Southern blot hybridization. As shown in Fig. 1B, all
A. chrysogenum and P. chrysogenum transformants with
the A. nidulans penDE gene formed IAT of molecular mass
39–40 kDa, whereas transformants carrying the P. chryso-

genum gene formed an IAT with a molecular mass of
28–29 kDa.
These results indicate that the A. nidulans penDE gene,
when expressed in P. chrysogenum or A. chrysogenum,
forms a protein that is not dissociated into its component
subunits, unlike the IAT of P. chrysogenum introduced into
the same host strains.
Constructions for expressing the
A. nidulans
IAT a
and b subunits and the ab proacyltransferase in
E. coli
As the 40-kDa proacyltransferase of P. chrysogenum cannot
be isolated as the undissociated protein because it is rapidly
self-processed in the cell, emphasis was put on the compar-
ative study of the activities of the A. nidulans 40-kDa
acyltransferase with those of its separate 29-kDa and
11-kDa subunits obtained by expressing the corresponding
DNA fragments in E. coli. To prepare constructions for
expression in E. coli, six different phages containing cDNA
for the A. nidulans penDE gene were isolated from about
30 000 phage plaques. The inserts in the five phages were
sequenced. None contained any of the three introns of the
penDE gene. Two of the cloned cDNA fragments
(pFCaat107 and pFCaat108) showed 5¢ ends that started
86 and 73 bp upstream, respectively, from the initial ATG
of the penDE ORF; two others (pFCaat104 and
pFCaat106) started 34 or 19 bp, respectively, downstream
from the ATG. The fifth phage (pFCaat100) contained an
insert that started 315 bp downstream from the ATG, close

to the nucleotide position 309–315, which encodes the CTT
motif corresponding to the processing site of the proacyl-
transferase into its subunits. The 3¢ termini of all cloned
cDNA inserts (except pFCaat108) extended past the PstI
site located 40 bp downstream from the TGA stop codon of
the penDE gene.
All inserts were recovered from the phagemids as EcoRI–
PstIfragments.TheEcoRI–PstI fragments were subcloned
into the pT7-7 vector for overexpression in E. coli.As
shown in Fig. 2, a translational fusion expression system
was constructed by subcloning the intronless form of penDE
gene as an SspI–PstI fragment from pFCaat107 into the
expression vector pT7.7. The resulting plasmid carried the
entire penDE gene (encoding the a and b subunits) in-frame
with a 13 amino-acid fragment of the pT7-7 system. To
avoid problems in the assay of IAT due to degradation of
isopenicillin N by the penicillinase encoded by the ampicillin
resistance gene of the pT7-7 system, the pT7-7-penDE
cassette was subcloned as a SmaI–NcoIfragmentinto
plasmid pBC KS+ (Stratagene) which contains the chlo-
ramphenicol (instead of ampicillin) resistance gene as
selective marker, thus obtaining plasmid pULCTaß. Three
other plasmids were constructed for overexpression in the
pT7-7 system: the a subunit (pULCTa), the b subunit
(pULCTb)andthea and b subunits in the same plasmid
but on separate ORFs (pULCTa+b). The different
constructions are shown in Fig. 2.
Two other similar constructions, pULCT106aß (carrying
both the a and b subunits as a single ORF, i.e. as they occur
in the proacyltransferase) and pULCT100b (with only the b

subunit), were constructed from the original inserts in phage
pFCaat106 and pFCaat100, respectively, by following
similar strategies. All constructions with the A. nidulans
intronless form of the penDE gene resulted in good
translation of the respective proteins (see below).
The 40-kDa
A. nidulans
proacyltransferase
is not processed
When the above constructions were introduced into E. coli
JM109(DE3), a strain containing the integrated T7 RNA
polymerase gene induced by IPTG, analysis of the proteins
by PAGE showed that a strong band of the A. nidulans
40-kDa protein is induced in E. coli transformed with
pULCTab and pULCT106ab, as expected. About 20% of
the total E. coli proteinwasestimatedtobeIATinextracts
of E. coli expressing these constructions (Fig. 3A, lanes
4and7).
Expression of the 40-kDa protein was also evidenced by
autoradiography of the labelled proteins after addition of a
pulse of [
35
S]methionine. Only two proteins, an unknown
25-kDa polypeptide (perhaps a fragment of the T7 RNA
polymerase induced by IPTG) and the 40-kDa IAT
(Fig. 3B, lanes 4 and 7), were labelled with methionine
under these expression conditions.
No self-processing of the 40-kDa A. nidulans proIAT
was observed in E. coli extracts. The IAT protein of E. coli
[pULCTab] always moved as a 40-kDa protein on SDS/

PAGE and no 29-kDa or 11-kDa subunits were observed
on either SDS/PAGE or autoradiography of the labelled
protein (a highly sensitive method). This result indicates that
the A. nidulans proIAT formed in E. coli is not self-
processed.
Similarly, the 29-kDa (b) subunit was overproduced in
E. coli transformedwithpULCTb after 1 and 3 min of
induction by IPTG (Fig. 3A, lanes 10 and 11). This protein
was clearly separated from the unknown 25-kDa polypep-
tide as shown in labelling experiments (Fig. 3B, lane 11).
Finally, the 11-kDa (a) subunit was also formed in E. coli
transformedwithpULCTa as shown by SDS/PAGE
(Fig. 3A, lanes 13 and 14) and by the labelling experiments
(Fig. 3B, lane 14).
Western blot analysis using the antibodies raised against
the A. nidulans 40-kDa proIAT confirmed these results
(Fig. 4). As shown in Fig. 4C, the 40-kDa proIAT is formed
at either 28 °Cor37 °Cbutnoa or b subunits were detected
in the immunoassays.
The
A. nidulans
40-kDa IAT obtained from
E. coli
shows acyltransferase activity
The E. coli [pULCTab] extracts containing unprocessed
A. nidulans proIAT showed acyltransferase activity
(Fig. 3C) [102 pmolÆmin
)1
Æ(mg protein)
)1

] equivalent to
that of the crude extracts of A. nidulans. The reaction
product was sensitive to b-lactamase and was identified as
penicillin G by HPLC.
The b subunit overexpressed in E. coli [pULCTb]did
not show IAT activity nor did the a subunit expressed
Ó FEBS 2003 Processing of the isopenicillin N acyltransferase (Eur. J. Biochem. 270) 1961
separately in E. coli [pULCTa]. No IAT activity was
observed when the extracts containing a and b subunits
were mixed together or when they were expressed from
plasmid pULCTa+b in which the genes encoding both
polypeptides are located (Fig. 3C). These results indicate
that in A. nidulans the ab 40-kDaIATistheactiveform,
whereas the mixture of a and b subunits expressed in E. coli
do not reconstitute to an active IAT.
A processed biologically active
P. chrysogenum
IAT
is recovered after expression in
E. coli
Similarly, recombinant phages carrying the P. chrysoge-
num intronless form of penDE gene were selected from the
cDNA library of P. chrysogenum by hybridization with a
1.6-kb XhoI–XbaI probe containing the penDE gene. The
absence of introns was confirmed by sequencing clones
containing fragments covering the intron sites; the 5¢ end
of the cDNA fragment was confirmed to be 37 nucleotides
upstream of the ATG translation initiation codon. Two
constructions, pPT7ab and pPBCab (Fig. 5), were assem-
bled in which the ORF of the acyltransferase was excised

with XmnI endonuclease at the AATG site (coinciding
with the ATG translation initiation codon) and linked to
the pT7-7 E. coli expression plasmid digested with NdeI,
filledwithdTTPandtreatedwithMung-beanexonuclease
to remove the protruding nucleotide. After the ligation,
the CATGCTT sequence at the linkage site was confirmed
by sequencing through the fusion point. In pPBCab the
chloramphenicol resistance gene was used as a marker
instead of the ampicillin resistance gene present in
pPT7ab.
As the unprocessed 40-kDa IAT was never recovered
from P. chrysogenum extracts, overexpression of the
P. chrysogenum penDE gene in E. coli was carried out
using constructions pPBCab and pPT7ab. As shown in
Fig. 2. Plasmids used to express the a and b subunits of the A. nidulans IAT in E. coli. Restriction endonuclease map of pULCTab,pULCTa,
pULCTb and pULCTa+b containing, respectively, the complete penDE gene of A. nidulans, the DNA fragments encoding the a or b subunits, or
the DNA regions corresponding to the a and b subunits on different fragments in the same plasmid. In all constructions the DNA fragments
encoding the penDE gene (or their fragments) were expressed from phage T7 promoter. Cm
R
, chloramphenicol resistance gene; Ap
R
, ampicillin
resistance gene; + F indicates filled ends after digestion with restriction endonucleases.
1962 F. J. Ferna
´
ndez et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Fig. 6, abundant expression of the 40-kDa ab protein was
obtained at 37 °CinE. coli transformed with each of
the constructions under induction conditions but not in
noninduced conditions (Fig. 6A).

The P. chrysogenum 40-kDa IAT could be easily recov-
ered as inclusion bodies in large amounts. The enzyme
purified in this manner showed no traces of the 29-kDa or
11-kDa polypeptides. However, when the pure 40-kDa
P. chrysogenum proIAT was solubilized and refolded
by dilution in a redox buffer containing poly(ethylene
glycol) and incubated for 6 h at room temperature, these
polypeptides were formed by autocatalytic cleavage
(Fig. 6C), and the heterodimeric form thus obtained
(containing the two subunits) showed IAT activity. Similar
results were reported by Tobin et al. [17].
To confirm that there were differences in proIAT
processing ability in the two fungi, constructions in E. coli
with either the P. chrysogenum or A. nidulans penDE genes
were expressed in parallel using [
35
S]methionine as marker.
The results (Fig. 7) clearly indicate that, whereas the 40-kDa
protein is rapidly processed in P. chrysogenum when
incubated at 28 °C (but not at 37 °C), the A. nidulans
remains as a 40-kDa protein when incubated at either
28 °Cor37°C.
The self-processing of the P. chrysogenum IAT was
confirmed by immunoblot studies using specific antibodies
to the P. chrysogenum IAT (Fig. 4). Western blot analysis
revealed that the P. chrysogenum 40-kDa proIAT is
efficiently self-processed when the gene is expressed at
28 °C but not at 37 °C, giving similar amounts of the
29-kDa and 11-kDa proteins (Fig. 4B, lane 2).
Extracts of

A. nidulans
do not process the 40-kDa IAT
obtained in
E. coli
To exclude the possibility that an A. nidulans peptidase
activity was required for in vivo processing, the labelled
40-kDa IAT obtained after expressing the A. nidulans
penDE gene in E. coli was incubated for 0, 30 and 60 min
with cell-free extracts of A. nidulans (7.92 mg proteinÆmL
)1
)
obtained from cells grown in penicillin production condi-
tions. The results showed that there is no processing of the
protein even after incubation for 60 min. The amount of
labelled protein remaining after treatment with the A. nidu-
lans extract was approximately the same as that observed
with boiled A. nidulans extracts.
Fig. 3. Proteins formed after expression of the A. nidu lans IAT a and b
subunits and assay of their catalytic activity. SDS/PAGE (A) and
autoradiography (B) of proteins expressed in E. coli from different
constructions with the penDE gene of A. nidulans.LaneM,molecular
massmarkers;lanes1,2,3,controlE. coli [pBC] without insert; lanes
4, 5, 6, E. coli [pULCTab];lanes7,8,9,E. coli [pULCT106ab];
lanes 10, 11, 12, E. coli [pULCTa]; lanes 13, 14, 15, E. coli [pULCTb].
Lanes1,4,7,10and13wereinducedwithIPTG.Lanes2,5,8,11and
14 were induced with IPTG and supplemented with a 15-min pulse of
[
35
S]methionine/[
35

S]cysteine. Lanes 3, 6, 9, 12 and 15 were not
induced. In the autoradiography, note the formation of a 40-kDa
labelled protein in lanes 5 and 8 (containing the ab constructions) and
proteins of 29 kDa in lane 11, and 11 kDa in lane 4 (containing,
respectively, the a or b subunits). A band of about 25 kDa observed in
all labelled preparations (lanes 2, 5, 8, 11 and 14) is an unknown
protein induced by IPTG. (C) Bioassay of the IAT activity using
extracts of E. coli transformed with different constructions with the
A. nidulans penDE gene. Formation of benzylpenicillin in the IAT
reaction was determined using Bacillus subtilis as test organism. 1,
E. coli JM109(DE3) [pULCTab] undiluted extract; 2, E. coli [pUL-
CTab]extractdiluted1:2;3,E. coli [pULCTb]; 4, E. coli [pULCTa];
5, control benzylpenicillin solution (1 lgÆmL
)1
); 6, same as 1 after
treatment of the extract with b-lactamase (Bactopenase; Difco); 7,
mixture of extracts of E. coli [pULCTa]andE. coli [pULCTb]; 8,
E. coli [pULCTa+b].
Ó FEBS 2003 Processing of the isopenicillin N acyltransferase (Eur. J. Biochem. 270) 1963
Immunoblot analysis of extracts of
P. chrysogenum
and
A. nidulans
shows different
in vivo
processing of the IAT
The availability of antibodies to IAT of P. chrysogenum
and A. nidulans allowed us to follow the in vivo processing
of IAT in both fungi. As shown in Fig. 8, Western blot
analysis of IAT in extracts of cells grown for 48, 72 or 96 h

revealed that P. chrysogenum IAT was already completely
processed to the 29-kDa (a) and 11-kDa (b) subunit at 48 h
and remained processed thereafter; the intensity of the
bands increased at 72 and 96 h, in agreement with the
enzyme being involved in secondary metabolism.
In contrast, the 40-kDa (unprocessed) IAT form (ab)was
clearly observed in A. nidulans cultures at 48, 72 and 96 h
and the intensity increased at 96 h. Degraded forms were
observed in the A. nidulans Western blot, but the 11-kDa
band was never observed, indicating that A. nidulans IAT is
processed or degraded differently from the P. chrysogenum
enzyme.
Discussion
Formation of mature enzymes from preproenzymes is a
common phenomenon in eukaryotic organisms. In most
cases, specific endopeptidases are involved in recognition
and cleavage of the proenzymes. Some proteins with pepti-
dase activity may process themselves autocatalytically [31].
The 40-kDa P. chrysogenum IAT is a heterodimer of a and
b subunits [17,18,32]. An important difference between the
IATs of P. chrysogenum and A. nidulans is that during
purification of the active form of the P. chrysogenum
enzyme, the 40-kDa heterodimer is never observed, and
instead the b-subunit (29 kDa) is enriched throughout the
purification process [20]. The significant loss of total enzyme
activity during purification of P. chrysogenum IAT is con-
sistent with the fact that only the 29-kDa protein is enriched
whereas both subunits are required for full enzyme activity.
When the penDE gene of A. nidulans was expressed in
IAT-deficient mutants of P. chrysogenum or in A. chryso-

genum, the molecular mass of the IAT formed was that of
Fig. 5. Restriction map of plasmids pPT7ab and pPBCab containing the pe nDE gene of P. chryso genu m under the T7 promoter. Plasmid pPT7ab
contains the ampicillin resistance marker whereas pPBCab contains the chloramphenicol resistance gene. Abbreviations are the same as in Fig. 3.
Fig. 4. Comparative expression of the genes pe nDE from P. chrysogenum and A. nidulans in E. coli at two different temperatures and immunodetection
of the IAT proteins. (A) SDS/PAGE of the cell lysates. (B) Immunological detection of the IAT protein from P. chrysogenum.(C)Immunological
detection of the IAT protein from A. nidulans. Lanes 1, 2, 8 and 9, E. coli [pPBCab], contains the gene penDE from P. chrysogenum (lanes 1 and 8
without IPTG induction and lanes 2 and 9 with 0.5 m
M
IPTG). Lanes 3, 4, 10 and 11, E. coli [pULCTab], contains the gene penDE from
A. nidulans (lanes 3 and 10 without IPTG induction and lanes 4 and 11 with 0.5 m
M
IPTG). Lane 5, molecular mass markers. Lanes 6 and 7, E. coli
[pT7.7] used as control (lane 6 without IPTG induction and lane 7 with 0.5 m
M
IPTG). Lanes 1–4 and 6–7 contain cell lysates obtained from
bacterial cultures grown at 28 °C, and lanes 8–11 contain cell lysates obtained from bacterial cultures grown at 37 °C.
1964 F. J. Ferna
´
ndez et al.(Eur. J. Biochem. 270) Ó FEBS 2003
the A. nidulans enzyme (40 kDa). All available information
supports the conclusion that self-processing is determined
by the amino-acid sequence of the IAT itself and not by the
host. Immunoblot studies using antibodies against P. chryso-
genum or A. nidulans IAT supported this conclusion. The
P. chrysogenum IAT was already fully processed after 48 h
of incubation under penicillin production conditions,
whereas the A. nidulans enzyme remained in the 40-kDa
form for at least 96 h of incubation.
No peptidases able to cleave IAT were found in
the fungal extracts. This indicates that processing of

P. chrysogenum IAT is autocatalytic and is consistent
with the observation of processed enzyme in the hetero-
logous Acremonium system when the P. chrysogenum
Fig. 6. Processing of the soluble form, inclusion bodies, and refolded forms of the IAT of P. chrysogenum. (A)SDS/PAGEofproteinsexpressedin
E. coli [pPBCab]andE. coli [pPT7ab] (containing the penDE gene of P. chrysogenum). Lane 1, noninduced E. coli [pPBCab]; lane 2, induced
E. coli [pPBCab]; lane 3, noninduced E. coli [pPT7ab]; lane 4, induced E. coli [pPT7ab]. Note the formation of the 40-kDa protein (arrow). (B)
SDS/PAGE of proteins collected as insoluble material (inclusion bodies) after overexpression in E. coli. Lane 1, molecular mass markers; lane 2,
total extract of E. coli [pPBCab]; lane 3, supernatant of E. coli [pBCab] extracts after centrifugation at 12 000 g; lane 4, insoluble material collected
from extracts of E. coli [pPBCab]; lane 5, total extract of E. coli [pPT7ab]; lane 6, supernatant of E. coli [pPT7ab]; lane 7, insoluble material of
E. coli [pPT7ab]. Note the presence in crude extracts and in the insoluble material of the 40-kDa protein (arrow). (C) Lane 1, proteins in the
inclusion bodies isolated from E. coli [pPBCab]; lane 2, inclusion bodies of E. coli [pPT7ab]; lane 3, size markers; lane 4, refolded proteins in the
inclusion bodies of E. coli [pPBCab]; lane 5, refolded proteins in the inclusion bodies of E. coli [pPT7ab]. Note that after refolding there is partial
processing of the 40-kDa protein into subunits a (11 kDa) and b (29 kDa) (arrows). The refolded proteins showed considerable IAT activity, which
was not detectable in the insoluble inclusion bodies.
Fig. 7. Comparative expression and processing in E. coli of the IATs encoded by the penDE genes of P. chrysogenum and A. nidulans at two different
temperatures. (A) SDS/PAGE of proteins. (B) Autoradiography of the gel. Lanes 1 and 2, E. coli [pPBCab]containingthepenDE gene of
P. chrysogenum without and with induction (note formation of the 29-kDa and 11-kDa subunits in lane 2); lanes 3 and 4, E. coli [pULCTab]
containing the penDE gene of A. nidulans without and with induction (note the formation of the 40-kDa protein and the lack of processing to the
29-kDa and 11-kDa subunits) in lane 4; lane 5, molecular mass markers; lanes 6 and 7, control E. coli [pT7-7] without and with induction; lanes 8
and 9, E. coli [pPBCab] without and with induction; lanes 10 and 11, E. coli [pULCTab] without and with induction. In lanes 1–7, cultures were
incubated at 28 °C, and in lanes 8–12 at 37 °C.
Ó FEBS 2003 Processing of the isopenicillin N acyltransferase (Eur. J. Biochem. 270) 1965
penDE gene was introduced into this fungus which lacks
IAT [4].
IAT of P. chrysogenum and A. nidulans catalyses the
third step of penicillin biosynthesis, namely the hydrolysis of
the peptide (amide) bond between a-aminoadipic acid and
cysteine of the penicillin nucleus (condensed cysteinyl-
valine) [33]. In this respect, IAT resembles cysteine pepti-
dases, and, indeed, the cleavage site of both P. chrysogenum

and A. nidulans IATs is the bond Gly102–Cys103 estab-
lished by the amino-acid sequence of the N-terminus of
the 29-kDa (b) subunit [6,32,33].
The IAT of P. chrysogenum is strongly inhibited by
phenylmethanesulfonyl fluoride [16], a well-known inhibitor
of serine proteases and acyltransferases. We proposed
previously that the GXS309XG motif is involved in
cleavage of phenylacetyl-CoA and binding of the phenyl-
acetyl moiety to the enzyme. Indeed, Tobin et al.[34]
reported that mutation of Ser309 to Ala abolished IAT
activity without affecting cleavage of the enzyme. These
authors have also shown that mutation of Ser227 alters
cleavage of the enzyme [17].
OurresultsindicatethatP. chrysogenum and A. nidulans
differ in their ability to self-process the IAT into a and b
subunits. This difference is unlikely to be due to amino-acid
sequences around the cleavage site because the sequence
99ARDG*CTT(V/A)YC, which includes the cleavage
site (indicated by an asterisk), is conserved in both
fungi, although the Val106 to Ala106 substitution in the
A. nidulans enzyme may have some effect on cleavage.
Similarly, Ser227 is conserved in both fungi, which indicates
that the inefficient processing and dissociation in A. nidulans
is not due to alteration of this particular residue (which may,
rather, be involved in isopenicillin N binding because it is
also conserved in several cephalosporin and cephamycin
biosynthetic enzymes [34]). However, the two IATs differ in
23.5% of their component amino acids (the two cysteines
included), which may explain their different autocatalytic
activities.

The lack of processing of the A. nidulans 40-kDa IAT
when expressed in soluble form in E. coli suggests that the
self-processing ability of IAT of A. nidulans is weak
compared with that of the P. chrysogenum enzyme. Twenty
additional amino acids were present in the E. coli-expressed
A. nidulans IAT. Although these amino acids may affect the
self-cleaving ability, they were not present in the construc-
tion used to transform the filamentous fungi (either
P. chrysogenum or A. chrysogenum), and the IATs formed
in the fungi remain undissociated, as occurs with the enzyme
formed in E. coli.The40-kDaA. nidulans IAT is enzymati-
cally active, whereas we did not observe any activity with
mixtures of the a and b subunits.
Penicillin acylases (amidases) occur in many micro-
organisms (reviewed in [35,36]). One of the best known,
the E. coli penicillin acylase, consists of two dissimilar
subunits derived from a membrane-bound single polypep-
tide precursor (proacylase) by autocatalytic processing
[37,38]. Autocatalytic processing usually leads to more
active forms, and this may explain the observed difference
in activity of the P. chrysogenum and A. nidulans IATs.
The similarity between the E. coli and fungal penicillin
amidases from this mechanistic point of view deserves
further studies.
P. chrysogenum and A. nidulans differ dramatically in
their ability to synthesize penicillin, although the gene
clusters are similar and occur in a single copy in the wild-
type strains of both fungi.
Crude extracts of wild-type P. chrysogenum strains show
about fivefold higher IAT activity than wild-type A. nidu-

lans strains (E. Montenegro and J.F. Martı
´
n, unpublished
results). It is possible that the difference in the ability to self-
process their respective IATs may affect the overall
penicillin-biosynthetic ability of the two fungi.
Fig. 8. Western blot analysis showing the different processing of the IATs of P. chrysogenum and A. nidulans. Extracts of P. chrysogenum AS-P-78
and A. nidulans ATCC 28901 grown in CP medium and MFA medium, respectively for 48, 72 or 96 h were obtained, resolved by SDS/PAGE, and
visualized with antibodies as described in Materials and methods. Lanes: M, molecular mass markers; 48, 48-h extracts; 72, 72-h extracts; 96, 96-h
extracts. The size of the molecular mass markers in kDa is shown on the left. The immunoreactive IAT bands of 40 kDa, 29 kDa and 11 kDa are
indicated by arrows on the right. Note the presence of the 40-kDa band and the absence of the 11-kDa IAT band in the A. nidulans extracts.
1966 F. J. Ferna
´
ndez et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Acknowledgements
This work was supported in part by grants from Antibio
´
ticos S.p.A.
(Milan, Italy) and the CICYT, Ministry of Education and Science
(BIO2000-1726-C02-01). F.J.F. and J.V. received fellowships from the
University of Leo
´
n. We thank F. Fierro for scientific discussions and
M. Corrales, M. Mediavilla and R. Barrientos for excellent technical
assistance.
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