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 precur
Trang 1The isopenicillin N acyltransferases of Aspergillus nidulans
the 40-kDa ab heterodimer in an undissociated form
Francisco J Ferna´ndez1, Rosa E Cardoza2, Eduardo Montenegro1, Javier Velasco1, Santiago Gutie´rrez1,2 and Juan F Martı´n1,2
1
Area de Microbiologı´a, Facultad de Ciencias Biolo´gicas y Ambientales, Universidad de Leo´n, Spain;2Instituto de Biotecnologı´a de Leo´n INBIOTEC, Parque Cientı´fico de Leo´n, Spain
The isopenicillin N acyltransferases (IATs) of Aspergillus
nidulansand 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 chrysogenumIAT 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 npe6 and npe8 or in
Acremo-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-lanscDNA 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 from a cDNA library was expressed in E coli, and the IAT 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 nidulansand Penicillium chrysogenum are able
to synthesize hydrophobic penicillins because of
substitu-tion of theL-a-aminoadipyl side chain of isopenicillin N by
aromatic acyl side chains catalyzed by the isopenicillin N
acyltransferase (IAT) [1–3], whereas Acremonium
chryso-genumlacks 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 are able to
synthesize 30-fold higher levels of penicillin than wild-type
A nidulans(their penicillin production levels are about 150 and 5 lgÆmL)1in 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: degjmm@unileon.es
Abbreviations: IAT, isopenicillin N acyltransferase; IPTG, isopropyl
thio-b- D -galactoside.
(Received 31 October 2002, revised 10 December 2002,
accepted 10 March 2003)
Trang 2there 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 nidulansproacyltransferases 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 coliexpressing 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) were u sed in the in 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 25C 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)1NaCl) at 4C,
suspended (420 g wet weight) in TD buffer (50 mM Tris/
HCl pH 8.0, 5 mMdithiothreitol) and disrupted with glass
beads in a mechanical cell disruptor (Braun, Melsungen,
Germany) under liquid CO2 refrigeration The extract
was centrifuged at 20 000 g for 20 min, and the
super-natant collected and used for further purification
Self-processing ofA 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 mM 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
The molecu lar mass of the IATs was established by gel 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 inE 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 37C u ntil an A600 of 0.4 was reached Expression of the penDE gene was induced by the addition of IPTG (final concentration 0.5 mM), 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 mM Tris buffer containing 5 mM EDTA, pH 5.0, and lysed by incubation with lysozyme (final concentration
1 mgÆmL)1) for 15 min at 4C followed by sonication The insoluble inclusion bodies were collected by centrifugation
at 12 000 g for 10 min and solubilized in 8Murea in redox buffer (containing 10 mM glutathione, 1 mM oxidised glutathione, 50 mMTris, 10 mMCaCl2and 10 mMMgCl2)
at pH 8.0
Proteins were refolded from solubilized inclusion bodies
by diluting the redox buffer to a final concentration of 4M urea and 100 lg proteinÆmL)1, and supplementing it with poly(ethylene glycol) 6000 [at a final molar ratio of poly(ethylene glycol) to protein of 10] [25]
Trang 3Preparation of antibodies against theP chrysogenum
orA 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 theA nidulans and P chrysogenum IAT
Gel-filtration chomatography on Sephadex G75 superfine
of the A nidulans IAT revealed a Kav 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 Kav 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 npe8 of P 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 chrysogenumor 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, and several 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 npe8 and P 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 npe8 and
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.
Trang 4contained one to three integrated intact copies of either the
P chrysogenumor 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 theA nidulans IAT a
and b subunits and the ab proacyltransferase inE 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–
PstI fragments The EcoRI–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–NcoI fragment into
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) and the a 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-kDaA 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 protein was estimated to be IAT in extracts
of E coli expressing these constructions (Fig 3A, lanes
4 and 7)
Expression of the 40-kDa protein was also evidenced by autoradiography of the labelled proteins after addition of a pulse of [35S]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 transformed with pULCTb 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 transformed with pULCTa 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 28C or 37 C bu t no a or b subunits were detected
in the immunoassays
TheA 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)1Æ(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
Trang 5separately 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-kDa IAT is the active form,
whereas the mixture of a and b subunits expressed in E coli
do not reconstitute to an active IAT
A processed biologically activeP chrysogenum IAT
is recovered after expression inE coli
Similarly, recombinant phages carrying the P
chrysoge-numintronless 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, filled with dTTP and treated with Mu ng-bean exonu clease
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 CmR, chloramphenicol resistance gene; ApR, ampicillin resistance gene; + F indicates filled ends after digestion with restriction endonucleases.
Trang 6Fig 6, abundant expression of the 40-kDa ab protein was
obtained at 37C in E 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 [35S]methionine as marker
The results (Fig 7) clearly indicate that, whereas the 40-kDa
protein is rapidly processed in P chrysogenum when
incubated at 28C (but not at 37 C), the A nidulans
remains as a 40-kDa protein when incubated at either
28C or 37 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
28C but not at 37 C, giving similar amounts of the
29-kDa and 11-kDa proteins (Fig 4B, lane 2)
Extracts ofA nidulans do not process the 40-kDa IAT obtained inE 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-lansextract was approximately the same as that observed with boiled A nidulans extracts
Fig 3 Proteins formed after expression of the A nidulans 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 Lane M, molecu lar
mass markers; lanes 1, 2, 3, control E coli [pBC] without insert; lanes
4, 5, 6, E coli [pULCTab]; lanes 7, 8, 9, E coli [pULCT106ab];
lanes 10, 11, 12, E coli [pULCTa]; lanes 13, 14, 15, E coli [pULCTb].
Lanes 1, 4, 7, 10 and 13 were indu ced with IPTG Lanes 2, 5, 8, 11 and
14 were induced with IPTG and supplemented with a 15-min pulse of
[35S]methionine/[35S]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] extract dilu ted 1 : 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] and E coli [pULCTb]; 8,
E coli [pULCTa+b].
Trang 7Immunoblot analysis of extracts ofP chrysogenum
andA 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 penDE gene of P chrysogenum 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 penDE 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) Immu nological 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.
Trang 8the 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-genumor A nidulans IAT supported this conclusion The
P chrysogenumIAT 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/PAGE of proteins expressed in
E coli [pPBCab] and E 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] containing the penDE 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.
Trang 9penDE 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]
Ou r resu lts indicate that P 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 nidulansIAT 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 chrysogenumor A chrysogenum), and the IATs formed
in the fungi remain undissociated, as occurs with the enzyme formed in E coli The 40-kDa A 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-lansstrains (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.
Trang 10This 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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