From the Insituto de Biotecnología,
Parque Científico de León, Avda. del Real no. 1,
León 24006 and the ¶ Universidad de León, Facultad de
Ciencias Biológicas y Ambientales, Area de Microbiología,
León 24071, Spain
Received for publication, November 4, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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The In fungi, lysine derives from In filamentous fungi, The domain structure of Penicillium chrysogenum Materials
L-1-Tosylamido-2-phenylethyl chloromethyl
ketone-treated bovine trypsin, elastase, thyoglycollic acid
(mercaptoacetic acid), and 3-cyclohexylamino-1-propanesulfonic acid
buffer were purchased from Sigma;
DL- Organisms and Culture Conditions
P. chrysogenum AMA, a strain derived from P. chrysogenum Wis 54-1255 pyrG, which overexpresses
lys2 in an autonomous replicating plasmid3 was used as Recombinant DNA Methods
Standard genetic techniques with Escherichia coli and
in vitro DNA manipulations were as described by Sambrook and
Russell (25). Polymerase chain reactions were performed using
Pfu DNA polymerase as described by the enzyme supplier
(Stratagene). DNA sequencing was accomplished by the dideoxynucleotide
chain-termination method using the PerkinElmer Life Sciences Amplitaq
Dye-terminator sequencing system on double-stranded DNA templates with
an Applied Biosystems model 310 sequencer (Foster City, CA).
Determination of Crude enzyme preparations were obtained by grinding the cells in
liquid nitrogen and suspending them in 10 mM Tris-HCl, pH 8.0. The extract was then dialyzed (cellulose membranes,
Mr 12,000) against the same buffer at 4 °C to
remove possible low molecular weight enzyme inhibitors. The Polyclonal Antibodies against Two fragments of Lys2 were chosen for the generation of
antibodies, one spanning from Gly302 to Pro763
(peptide A) and the second one from Gly402 to
Ser1292 (peptide B). The DNA encoding both
fragments was amplified using PCR methods from plasmid pBL2, which
contains the lys2 gene from P. chrysogenum
(15), using the following primers: 1)
5'-ACGAATTCATGGCATTCAGCGGGGCGAG-3', 2)
5'-ACGAATTCAGGTACAAAGTAGCTGAC-3', and 3)
5'-ACGAATTCGGAAGTAGACCAAGGCAC-3' (EcoRI site
underlined). The PCR product resulting from amplification with primers
1 and 2 (1383 bp) was digested with EcoRI and cloned into
pGEX-2T to give pGEXA. Similarly, pGEXB was obtained by cloning of the
BamHI-EcoRI-digested PCR product resulting from
amplification with primers 1 and 3 (2988 bp) into pGEX-2T. Both
plasmids were used to transform E. coli BL21 cells.
Cultures of E. coli BL21 transformants were grown at
30 °C in 2× TY medium with 100 µg/ml ampicillin to
A600 of 1.0 and then induced with 0.2 mM isopropyl 1-thio- Peptides A and B were eluted from polyacrylamide gels and used to raise
antibodies as follows: 100 µg of peptide in 500 µl of PBS was
emulsified with 500 µl of Freund's adjuvant (Difco Laboratories
Inc.), and the emulsion was used to immunize New Zealand White
rabbits by intradermal injection. This process was repeated
three times, every 2 weeks, using incomplete Freund's adjuvant. After
immunization was complete, blood serum was collected by centrifugation
and the IgG fraction was purified by ammonium sulfate precipitation,
Sephadex G-25 desalting, and fast-protein liquid chromatography using a
protein A-Sepharose HR 10/2 column as previously described (27).
Preparation of an Immunoaffinity Column with Anti- The Affi-Gel Hz (Bio-Rad Laboratories) system was used for the
preparation of an immunoaffinity column. Binding of the anti- Enzyme Purification
All purification procedures were performed at 4 °C. P. chrysogenum mycelium (40 g of wet weight) was washed and ground in liquid nitrogen. The disrupted mycelium was resuspended in buffer A (25 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 1 mM 1,4-dithiothreitol) and ultracentrifuged at 20,000 × g for 1 h. The supernatant was then used as the
source of enzyme.
Step 1: Anion-exchange Chromatography--
The mycelium extract
(2085 mg of protein and 5525 units of enzyme) was applied to a
DEAE-Sepharose Fast Flow column (3.4 × 11 cm, 100 ml)
equilibrated with buffer A (flow rate, 1.2 ml/min). The column was
washed with 200 ml of this buffer before elution with a 600 ml of
linear gradient of 0-0.5 M NaCl in buffer A. Fractions (6 ml) were collected, and those enriched in Step 2: Sephacryl S-300 Chromatography--
The DEAE-Sepharose
pool was concentrated to 4 ml in Centriprep YM-50 concentrators
(Millipore), diluted 20-fold in buffer A containing 75 mM
NaCl, and again concentrated to 4 ml. This preparation was then applied
(flow rate, 1.4 ml/min) to a Sephacryl S-300 column (2.6 × 90 cm;
478 ml) equilibrated with buffer A containing 75 mM
NaCl. Fractions (7 ml) were collected and assessed for the
presence of Step 3: Phenyl-Sepharose Chromatography--
The Sephacryl S-300
pool (22 ml, 23 mg of protein, 1620 units) was diluted 3-fold in buffer
A containing 1.5 M
(NH4)2SO4 (to achieve a final
concentration of 1 M
(NH4)2SO4 and applied (flow rate
1.1 ml/min) to a phenyl-Sepharose 6 Fast Flow column (1.5 × 6.4 cm; 11 ml) previously equilibrated with the buffer A containing 1 M (NH4)2SO4. Bound
proteins were eluted with a linear gradient of 1 to 0 M
(NH4)2SO4 in buffer A, and
subsequently the enzyme was eluted with H2O. The final
volume of the pooled active fractions was 50 ml (2 mg of protein, 339 units).
Step 4: Immunoaffinity Chromatography--
The phenyl-Sepharose
pool was dissolved in buffer A and applied (flow rate 0.3 ml/min) to an
immunoaffinity column (5 ml; 5.67 mg of IgG anti- Limited Proteolysis of Purified Acylation of Labeling was performed at 30 °C, before or after limited
proteolysis, using DL- Analytical Methods
Protein concentrations were estimated using the Bradford method
(28) or by measuring the absorbance of column eluates at 280 nm.
SDS-PAGE was performed in 7, 10, or 12% polyacrylamide gels by
standard procedures. After electrophoresis, gels were stained either
with Coomassie Brilliant Blue R-250 or with silver. Alternatively gels
were transferred to ProBlott polyvinylidene difluoride membranes
according to Matsudaira (29) and subjected to Edman degradation on an
Applied Biosystems 477A pulsed-liquid protein sequencer. The Purification of P. chrysogenum
The molecular mass of the enzyme was estimated to be ~155 kDa by
SDS-PAGE (Fig. 1), which is in close agreement with the predicted value
deduced from the sequence (154.8 kDa). Molecular mass determination of
the enzyme on Superose 6 under non-denaturing conditions yielded a mass
of 171 ± 11 kDa, whereas analysis on Superose 12 resulted in a
value of 169 ± 10 kDa. Taken together, these results suggest that
the native enzyme has a monomeric nature.
Optimal Conditions for Activity--
In a standard 60-min reaction
the optimal temperature was found to be in the range of 25-29 °C.
Above 30 °C the enzyme activity was rapidly lost. Within this
temperature range, the period of linear phase of reaction lasted for 30 min, and the enzyme remained active for at least 2 h.
The
The pH dependence of enzyme activity on
The analysis of the effect of divalent cations on activity showed that
Mg2+ is absolutely required for activity; the optimal
concentration was 10 mM. Mn2+ could replace
Mg2+ to some extent (optimal concentration 5 mM), yielding 60% of the maximum activity attained with
Mg2+. The ions Ca2+, Co2+,
Hg2+, and Cu2+ could not substitute for
Mg2+. The addition of chelating agents such as EDTA (5 mM) to the reaction mixture produced a 75% inhibition of
the activity, further supporting the requirement of Mg2+
for the reaction.
Kinetics of Substrate Specificity of the Enzyme--
Several analogs of the
Lys2 substrate, Limited Proteolysis of
The fragmentation pattern generated by tryptic or elastase cleavage was
similar. Fig. 2 shows the fragment pattern generated by trypsin. The
initial cleavage introduced by trypsin resulted in the release of a
116-kDa fragment (T1) and a 28-kDa fragment (T2) as the first stable
products (see Fig. 2B). The size on SDS-PAGE and the
N-terminal sequence of T1 showed that it comprises the adenylation
domain, the PCP, and the NADPH binding site (19). Similarly, the
N-terminal sequence of T2 indicated that this fragment includes most of
the reduction domain, excluding the nucleotide binding site. As the
hydrolysis continued, the C-terminal piece of T1 was removed leading to
the generation of a 105-kDa fragment (T3) containing the adenylation
and PCP domains. Subsequently, this fragment losses its N-terminal end
resulting in a 76-kDa protein fragment (T4).
As the time of incubation was increased, further fragments were
generated concurrent with the disappearance of T3 fragment. Fragments
T5 (30 kDa) and T6 (64 kDa) have the same N terminus as T3, and both
have lost the PCP domain. Finally, other fragments generated were T7
(32 kDa), T8 (30 kDa), and T9 (29 kDa), which contain the C-terminal
end of the adenylation domain (Fig. 2B). T8 also contains
the whole PCP domain (see below). Under harsher conditions, the
fragments become unstructured and get degraded.
As summarized in Fig. 2, the primary cleavage site is located at the
N-terminal portion of the reductive domain (N-terminal end of fragment
T2 (Ile1141)), dividing the molecule into two independent
parts, namely the di-domain comprising the adenylating and PCP domains,
including the NADPH-binding region located C-terminal of the PCP, and a fragment containing most of the reductive domain excluding the NADPH-binding site.
Time-dependent Acylation of
To determine which domains were being acylated, various proteolytic
digests of
These results were corroborated by liquid scintillation counting of
peptide-bound radioactivity in slices of the polyacrylamide gel (see
"Experimental Procedures"). Again, no significant difference was
found between labeling before or after proteolysis, indicating that the
adenylation domain functions as an independent entity and that there is
no need for collaboration with other domains to load its substrate.
Interestingly, some level of acylation could be detected in the
PCP-containing fragment (T8) by using the gel slice method regardless
of whether labeling was made before or after proteolysis. No labeled T8
protein band could be detected in the fluorography assay possibly due
to low sensitivity of fluorography for tritiated compounds.
Acid-mediated Release of
Very similar results were obtained after treatment with formic or
performic acid of a mixture of the labeled fragments obtained after
limited tryptic digestion. These results clearly indicate that the
This report represents the first purification to homogeneity of
any natural The purification of P. chrysogenum The relatively high Km value of the purified enzyme
for The purification to homogeneity of the Proteolytic studies on The remaining fragments identified belong to different regions of the
adenylation domain, including or not the PCP domain. The latter was
never found as an independent fragment, which is not unusual, because,
in other multifunctional proteins harboring PCP or ACP domains like
non-ribosomal peptide synthetases or polyketide synthases, it is also
associated to adjacent domains (35, 39, 40).
Interestingly, fragment T5 corresponds to the N-terminal portion of the
Lys2 protein (~270 residues), a region presently unassigned for
function. Data base searches with this region revealed a significant degree of identity with DUF4 domains of nostopeptolide synthases (NosA proteins), other Lys2 proteins, peptide synthetases
(e.g. P. chrysogenum ACV synthetase,
Bacillus subtilis surfactin synthetase 2 and 3, and
Bacillus brevis gramicidin synthetase) or CoA ligases, among
others. The function of this D4F4 domain remains unknown, although its
conservation in a wide range of multifunctional proteins suggests a
structural role, perhaps in maintaining the integrity of the protein.
Acylation of Formation of thioester intermediates has been shown also in the
activation of phenylalanine in the tyrocidine synthase 1 of B. brevis (40). This enzyme catalyzes the activation,
thioesterification, and epimerization of the
D-phenylalanine component of tyrocidine. The same mechanism
occurs therefore in The finding that, after trypsin digestion, all fragments containing
both the adenylation active site and the PCP domain (T1, T3, and T4)
were labeled by The residual labeling observed in fragment T8, which comprises
the PCP domain but lacks the adenylation box, occurred regardless whether labeling was previous to the proteolysis or not, suggesting either that the PCP domain suffers direct loading of
-aminoadipate reductase (
-AAR) of
Penicillium chrysogenum, an enzyme that activates the
-aminoadipic acid by forming an
-aminoadipyl adenylate and
reduces the activated intermediate to
-aminoadipic semialdehyde, was
purified to homogeneity by immunoaffinity techniques, and the kinetics
for
-aminoadipic acid, ATP, and NADPH were determined. Sequencing of
the N-terminal end confirmed the 10 first amino acids deduced from the
nucleotide sequence. Its domain structure has been investigated using
limited proteolysis and active site labeling. Trypsin and elastase were used to cleave the multienzyme, and the location of fragments within
the primary structure was established by N-terminal sequence analysis.
Initial proteolysis generated two fragments: an N-terminal fragment
housing the adenylation and the peptidyl carrier protein (PCP) domains
(116 kDa) and a second fragment containing most of the reductive domain
(28 kDa). Under harsher conditions the adenylation domain (about 64 kDa) and the PCP domain (30 kDa) become separated.
Time-dependent acylation of
-AAR and of fragments containing the adenylation domain with tritiated
-aminoadipate occurred in vitro in the absence of NADPH. Addition of
NADPH to the labeled
-AAR released most of the radioactive
substrate. A fragment containing the adenylation domain was labeled
even in absence of the PCP box. The labeling of this fragment (lacking PCP) was always weaker than that observed in the di-domain (adenylating and PCP) fragment suggesting that the PCP domain plays a role in the
stability of the acyl intermediate. Low intensity direct acylation of
the PCP box has also been observed. A domain structure of this
multienzyme is proposed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ketoglutarate via the so-called
-aminoadipate (
-AA)1
pathway (1, 2), a biosynthetic route that starts with the condensation
of acetyl-CoA and
-ketoglutarate to form homocitrate (3), which
is later subjected to isomerization
(4),2 oxidative
decarboxylation and amination to yield
-AA. This intermediate is
then converted into
-AA-
-semialdehyde by the action of the
-aminoadipate reductase (
-AAR, EC 1.2.1.31) encoded by the lys2 and lys5 genes. The product of the
lys2 gene constitutes the apoenzyme, whereas the
lys5 product appears to be a specific phosphopantetheinyl
transferase for post-translational modification of Lys2 (5, 6). The
-AAR, also called
-aminoadipate semialdehyde dehydrogenase, first
activates the
-AA
-carboxyl group by an ATP-dependent
process through the formation of an
-AA-adenylate, a unique step
among amino acid biosynthetic pathways, that is then reduced by the
reduction domain using NADPH to yield
-AA-
-semialdehyde and AMP.
-AA is not only an essential intermediate in
lysine biosynthesis but also a well-known precursor in the biosynthesis
of
-lactam antibiotics (7-9). It constitutes the branching point
for lysine and penicillin or cephalosporin biosynthesis, where it is
condensed with L-valine and L-cysteine to
form the tripeptide
-L-(
-aminoadipyl)-L-cysteinyl-D-valine (ACV) by the ACV synthetase (10-12) a member of the large family of
non-ribosomal peptide synthetases (13, 14).
-AAR has
been predicted (15) on the basis of sequence comparison analyses with
other
-AARs (16-19). The
-AAR also shows a striking similarity with non-ribosomal peptide synthetases in the N-terminal two-thirds of
the protein and NAD-dependent dehydrogenases in the last
third of the enzyme. However, direct evidence for the topology and
domain structure of native
-AAR enzymes has never been achieved, in part due to the lack of efficient purification protocols. Some purification attempts have been described for Saccharomyces
cerevisiae (20) and P. chrysogenum (21) native enzymes;
however, purification to homogeneity has never been previously
attained. Limited proteolysis (22, 23) has been used in this report, in
combination with specific active site radiolabeling, to study the
native
-AAR of P. chrysogenum and to provide the first
structural information on any fungal
-AAR. This work provides
structural and functional evidence of the domains of the
-AAR in
comparison with the domains occurring in the
-AA activating domains
of the five known ACV synthetases.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-[3H]aminoadipic acid (38 Ci
mmol
1) was synthesized by Amersham Biosciences. The
expression vector pGEX-2T and Sephadex G-25, protein A-Sepharose,
phenyl-Sepharose, Sephacryl S-300, DEAE-Sepharose, Superose 6, and
Superose 12 columns and high range molecular weight standards for
SDS-PAGE were from Amersham Biosciences; the immunoaffinity column was
prepared using the Affi-Gel Hz system (Bio-Rad Laboratories); ProBlott
polyvinylidene difluoride membranes and sequencing reagents were from
Applied Biosystems Inc. All other reagents were from commercial sources and of analytical grade.
-AAR
source to purify this enzyme. Sporulation in PW medium and growth in
DPM medium was performed as described elsewhere (24).
-AAR Activity
-AAR
activity was assessed by the procedure of Sagisaka and Shimura (26), as
described by Suvarna et al. (18). The standard reaction
mixture contained 12.5 mM
-AA , 15 mM ATP,
10 mM MgCl2, 1 mM reduced
glutathione, 0.625 mM
-NADPH, 250 mM
Tris-HCl, pH 9.0, and an appropriate amount of enzyme solution, in a
total volume of 1 ml. Reaction mixtures lacking
-AA were used as
controls. Reactions were incubated at 30 °C for up to 1 h and
terminated by the addition of 1 ml of 2%
p-dimethylaminobenzaldehyde in 2-methoxyethanol. The
enzyme activity was determined by quantifying the formation of
-AA-
-semialdehyde as previously described (19). The
-AA-
-semialdehyde forms a yellow complex with
p-dimethylaminobenzaldehyde (
max = 460 nm).
One unit of
-AAR is defined as the activity that gives an increase
of absorbance at 460 nm of 0.01 per min.
-AAR
-D-galactoside and grown for an additional 2 h. Cells were resuspended in
phosphate-buffered saline (PBS) and lysed by sonication using an
ultrasonic processor XL apparatus (Misonix Inc.). When formation of
inclusion bodies occurred, the inclusion bodies were isolated by low
speed centrifugation (3000 × g; Sorvall GSA rotor, 5 min), the precipitate was washed twice with PBS, and the inclusion
bodies precipitate was dissolved in 10% SDS. Digestion of the
resulting glutathione S-transferase (from Schistosoma
japonicum) fusion proteins was accomplished with thrombin (500 units/mg of protein, at room temperature for 4 h), and the
products were resolved by SDS-PAGE.
-AAR
Antibodies
-AAR antibodies to the matrix was performed according to the supplier's instructions. Purified IgG (12.6 mg) was used to react with 5 ml of the
matrix yielding 45% efficiency in the binding reaction.
-AAR were combined (60 ml)
and designated as the DEAE-Sepharose
-AAR pool (96.5 mg of protein,
5418 units).
-AAR by SDS-PAGE and immunodetection.
-AAR) previously
equilibrated with buffer A. The column was washed with 5 volumes of
this buffer containing 0.5 M NaCl and with 5 volumes of
buffer A before elution with a solution of 100 mM
glycine-HCl buffer, pH 2.5. Immediately after elution, 100 µl of 1 M Tris-HCl, pH 8.0, was added to each fraction (1 ml)
containing the enzyme to avoid protein denaturation. The enzyme
solution was concentrated in a Centriprep YM-50 concentrator, dialyzed
against buffer A containing 50% (v/v) glycerol, and preserved at
20 °C.
-AAR
-AAR was incubated with
L-1-tosylamido-2-phenylethylchloromethyl ketone-treated
trypsin at an enzyme/substrate ratio of 1/20 (w/w) or with elastase at
an elastase/substrate ratio of 1/10 (w/w) in distilled water. Reactions
were performed at 30 °C for various times and terminated by heating
at 100 °C for 5 min in electrophoresis sample buffer (unless
otherwise indicated). The reaction products were separated by SDS-PAGE,
and after electrophoresis, gels were stained either with Coomassie
Brilliant Blue R-250 or transferred to ProBlott membranes for
N-terminal sequencing.
-AAR with Labeled
DL-
-[3H]Aminoadipic Acid
-[3H]aminoadipic
acid in the absence of NADPH. Reactions (25 µl) contained 250 mM Tris-HCl, pH 9.0, 21 mM MgCl2,
32 mM ATP, 2.2 mM reduced glutathione, 11.4 µM DL-
-[3H]AA, and 1.6 µg
-AAR. The preparations were incubated for 10 min, terminated by
rapid freezing on dry ice/acetone, and stored at
80 °C. The
trichloroacetic acid precipitation assay was performed with 10%
trichloroacetic acid in the presence of 200 µg of bovine serum
albumin. Precipitated proteins were washed twice with 5% trichloroacetic acid and resuspended in 100 µl of 1 M
Tris base, and the amount of incorporated
DL-
-[3H]AA was quantified by liquid
scintillation counting. For fluorography, gels were soaked for 30 min
in 1 M sodium salicylate (pH 6.0) solution and dried before
exposure to Hyperfilm-MP (Amersham Biosciences); otherwise the
radioimmunoassay for determination of peptide acylation involved gel
slicing, protein extraction with 30% H2O2 (3 h
at 60 °C), and 3H-label quantification by liquid
scintillation counting.
-AAR
molecular mass was determined by microfast protein liquid
chromatography using a SMART system (Amersham Biosciences) and a
Superose 6 or Superose 12 column previously equilibrated with buffer A
containing 75 mM NaCl. Chromatography was performed at a
flow rate of 50 µl/min at 4 °C. Superose 6 and Superose 12 columns
were calibrated for molecular mass determination using the following
proteins: thyroglobulin, ferritin, catalase, aldolase, and bovine serum albumin.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-AAR and Determination of the
N-terminal Sequence--
The enzyme was purified from mycelium grown
for 24 h (early phase of growth) in DPM medium. A summary of the
specific activity and recovery of the enzyme during the purification
procedure is given in Table I. Each
purification step was also analyzed by SDS-PAGE followed by silver
staining (Fig. 1). The purified enzyme showed a homogenous protein band on SDS-PAGE. The enzyme was purified 117-fold with a yield of 5.5% from the cell extract. The purified enzyme rapidly lost activity, even in 50% (v/v) glycerol at
20 °C, probably due to denaturation and degradation of this large protein as deduced by immunoblot analysis of old preparations (not
shown). The N-terminal sequence of the
-AAR protein obtained from
the immunoaffinity column was Met-Ala-Val-Gly-Thr-Ala-Ser-Leu-Gln-Asp, which fully agrees with the sequence deduced from the lys2
gene starting at the first ATG codon of the open reading frame
(15).
Purification of P. chrysogenum -AAR
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Fig. 1.
Purification of the
-aminoadipate reductase of P. chrysogenum. A, SDS-PAGE (8%
polyacrylamide) of cell extracts and partially purified fractions.
Lanes: M, molecular weight markers: myosin (212 kDa),
2-macroglobulin (170 kDa),
-galactosidase (116 kDa),
transferrin (76 kDa), glutamate dehydrogenase (53 kDa). 1,
cell free extract; 2, active fractions after DEAE-Sepharose
anion exchange; 3, active fractions after Sephacryl S-300
gel filtration; 4, active fractions after hydrophobic
interaction chromatography; 5, active fractions after
immunoaffinity chromatography. Note the enrichment of the 155-kDa
protein band arrow. B, lane 1,
immunodetection (Western blot) of
-aminoadipate reductase of the
sample in lane 5.
-AAR stability was assessed by measuring activity after a
previous treatment (5-60 min) at
20 °C, 4 °C, 30 °C, or
37 °C. Whereas at
20 °C or 4 °C the enzyme remained stable
during the whole treatment, and at 30 °C there was a 20% loss in
activity after 1 h of incubation. This activity loss was
particularly evident at 37 °C where only 50% activity remained
after 15 min of incubation.
-AA was measured in 100 or
250 mM MES-NaOH, pH 5.5-7.5, 100 or 250 mM
Tris-HCl, pH 7.0-9.0, and 100 or 250 mM glycine-NaOH, pH
8.5-11.0, buffers. The enzyme activity was favored by using buffers
with the higher ionic strength and became apparent throughout the pH
range 7.5-9.5 with a peak at pH 9.0 (not shown).
-AA Conversion to
-AA-
-Semialdehyde--
The
formation of
-AA-
-semialdehyde by reduction of
-AA was
monitored spectrophotometrically by measurement of the cyclized form of
-AA-
-semialdehyde,
1-piperidine carboxylate, which reacts with
p-dimethylaminobenzaldehyde giving a yellow complex (
max = 460 nm). Using this assay, the
Km of P. chrysogenum Lys2 for its
substrate
-AA was determined to be 1.4 ± 0.1 mM, and the kcat was 66 ± 5 min
1. In a similar way, the Km for ATP
was 1.3 ± 0.0 mM, whereas for NADPH it was 160 ± 5 µM.
-AA, were assayed in the reaction mixture at the
same concentration, including adipic acid,
DL-diaminopimelic acid, L-glutamate, and
S-carboxymethyl-L-cysteine. Only
S-carboxymethyl-L-cysteine could be
accepted as substrate by P. chrysogenum
-AAR, which
yielded, however, 20-fold lower activity than with its natural substrate.
-AAR Generated Fragments Containing the
Adenylation and the Different Domains--
Lys2 was digested with
either trypsin or elastase at various molar ratios at 30 °C as
described under "Experimental Procedures" for various lengths of
time, and the resulting fragmentation pattern is shown in Fig.
2. Fragment patterns were unaltered when
SDS-PAGE was performed in the presence of dithiothreitol or
2-mercaptoethanol. Limited proteolysis was also attempted using
chymotrypsin, but no stable fragments were observed.
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Fig. 2.
Predicted domain organization and tryptic
fragmentation pattern of the P. chrysogenum
-aminoadipate reductase. A,
conserved motifs in the
-aminoadipate reductases (numbers
1-12; see Refs. 15 and 19 for the amino acid sequences of the 12 motifs) and organization of the predicted adenylation, PCP, and
reduction domains of this protein. Motif number 10 corresponds to the sequence LGGHSI containing the serine to which the
phosphopantetheine acid is bound (PCP domain). B, limited
proteolysis fragments (T1 to T9) of the
-aminoadipate reductase obtained by increasing the duration of the
tryptic treatment. The determined N-terminal amino acid sequence of
each tryptic fragment is indicated on its left end. The
number before the amino acid sequence indicates the position of the
first amino acid of each fragment in the protein.
-AAR Occurs in the
Absence of NADPH, and the Label Is Released by Cofactor
Addition--
A trichloroacetic acid-mediated protein precipitation
assay was used to assess the DL-
-[3H]AA
time-course acylation of P. chrysogenum
-AAR in the
absence of NADPH (Fig. 3). Under such
conditions the aminoacyl adenylate cannot be reduced and remains bound
to the enzyme (5). NADPH addition after 10 min of incubation released
most of the radioactivity, indicating that in the presence of NADPH the
full reaction is resumed, which is consistent with the reductive
cleavage of the
-AA-S-PCP acyl enzyme to form the
-AA-
-semialdehyde.
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Fig. 3.
Time-dependent acylation and
release of the labeled substrate following NADPH addition. Pure
-AAR was labeled by incubation with uniformly labeled
-[3H]AA (black circles). After 10 min of
incubation NADPH (0.62 mM) was added (vertical
arrow) to the reaction mixture, and the protein-bound label was
quantified as described under "Experimental Procedures." Note the
rapid release of label following NADPH addition (black
triangles) (see text for details).
-AAR Fragments Containing the Adenylation Domain Were Labeled
Even in the Absence of the PCP Box--
When the
-AAR protein was
labeled in the absence of NADPH and then subjected to SDS-PAGE and
fluorography, the
-[3H]AA radioactivity was found
associated with the Lys2 polypeptide (Fig.
4B, lane 1).
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Fig. 4.
Acylation of the
-AAR with
-[3H]AA before or after limited
proteolysis. A, limited proteolysis fragments
(T0-T9) of
-AAR (4 µg) obtained after increasing times
of trypsin (200 ng) treatment. Lanes 1 and 2,
molecular mass markers sizes (in kDa) are indicated on the
left; lane 3, undigested
-AAR; lanes
4 and 5, protein fragments obtained after tryptic
digestion for 10 or 30 min, respectively. B, fluorography
showing the binding of labeled
-[3H]AA to the
-AAR
fragments. Lane 3, labeled undigested protein; lanes
4 and 5, labeled
-AAR protein digested for 10 or 30 min, respectively; lane 6, labeling with
-[3H]AA of fragments obtained by previous digestion of
-AAR for 30 min. Note that the results of labeling before or after
tryptic digestion of
-AAR are similar (lanes 5 and
6).
-AAR were labeled as indicated above. Alternatively, the
protein was first labeled and subsequently proteolyzed. In both cases,
the pattern of labeled bands was identical (Fig. 4), finding
radioactivity bound to fragments T1, T3, T4, and T6 but not to other
small fragments. This indicates that any fragment containing the
adenylation box (Fig. 2A) (30) can be labeled. The labeling
observed in T6 is significant, because this fragment lacks the PCP
domain. This labeling was always weaker than that obtained in other
fragments (see below), indicating that the PCP domain might play a role
in the stability of the acyl intermediate.
-[3H]AA from Acylated
-AAR Shows That It Is Bound through a Thioester
Linkage--
Binding of the
-AA to the
-AAR following its
activation as the
-aminoadipyladenylate has been proposed to occur
through a thioester bond with the SH group of the
phosphopantetheine arm (Fig. 5), although
the formation of a direct ester group with a serine or threonine
hydroxyl group of the enzyme can not be excluded. The nature of the
bond formed can be differentiated by the selective hydrolysis of ester
bonds with formic acid, whereas formic acid treatment does not cleave
thioester bonds that require a treatment with performic acid (31).
Treatment of the acylated enzyme (in the absence of NADPH) with formic
did not release
-AA, whereas a clear release of
-[3H]AA, as observed by thin layer chromatography of
the cleavage reaction product after protein precipitation, was obtained
by treatment with performic acid (not shown).
View larger version (13K):
[in a new window]
Fig. 5.
Domain structure model of the
-AAR protein and proposed mechanism of substrate
activation and reduction. A, adenylation domain.
PCP, peptidyl carrier protein; R, reduction
domain; N, NADPH binding site; DUF, unknown
function domain (see text for details).
-AA molecule in the absence of NADPH remains bound as a thioester to
the phosphopantetheine arm of the PCP domain rather than to a serine or
threonine residue in the
-AA reductase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-AAR. Some previous attempts for purification of
-AAR
have been described for the S. cerevisiae enzyme (20) and
for the P. chrysogenum enzyme (21, 32), but in both cases the purification protocols yielded enzyme preparations only partially pure. Recently, the Lys2 enzyme has been cloned from S. cerevisiae DNA, overexpressed in E. coli, and purified
(5).
-AAR has been greatly
facilitated by (i) the use of mycelium from the strain P. chrysogenum AMA, a strain that shows
-AAR yields 10-fold higher
than its parental strain P. chrysogenum Wis
54-12553 and (ii) obtaining anti-Lys2 antibodies, which
turned out to be highly specific for P. chrysogenum
-AAR
(24), thus allowing the use of an efficient immunoaffinity column. The
native enzyme has been shown to be a monomer, and its N terminus
corresponds exactly to that proposed from the cloned gene (15).
-AA might be the reflection of a metabolic bias toward
penicillin biosynthesis in the strain used for enzyme purification. The
strain used derives from P. chrysogenum Wis 54-1255, which
shows partial lysine requirement in minimal medium and an improved
penicillin production when compared with the wild
type.4 The relatively low
affinity of
-AAR toward its substrate
-AA would lead to an
increase in the
-AA intracellular levels, which favor the formation
of the tripeptide
-L-(
-aminoadipyl)-L-cysteinyl-D-valine, and hence penicillin formation (21). We have previously correlated high
penicillin-producing strains with increased intracellular
-AA pools
(15). The low affinity of
-AAR toward
-AA may, therefore, direct
the
-AA pool to ACV formation.
-AAR from P. chrysogenum has provided the first opportunity to explore the
structure of this multifunctional key enzyme for lysine and
-lactam
biosynthesis. In general, multifunctional proteins are composed of
independently folded, compact domains connected by linker regions (33,
34), which are readily cleavable by limited treatment with proteolytic enzymes (35). From x-ray diffraction studies, it has been demonstrated (36) that segments of the polypeptide chain of high flexibility are
correlated with those regions that are vulnerable to limited proteolysis. Many interdomain loops are located at the surface of the
protein and therefore adopt accessible conformations that facilitate
their interaction with proteases.
-AAR were performed using two different
proteinases, chosen for their relatively broad primary specificity, so
that the accessibility to potential target sites, rather than the
specificity of the protease itself, is the factor likely to determine
the sites of cleavage. The similar pattern of digestion obtained with
both proteases reassures this hypothesis. N-terminal sequencing of the
fragments generated allowed them to be placed within the context of the
known primary structure of Lys2 (15). The initial cleavages observed
indicated that the multifunctional protein (155 kDa) could be divided
into two fragments of 116 and 28 kDa as estimated by SDS-PAGE, with a
cutting site at Arg1140-Ile1141. The large
fragment comprises the adenylation domain, the PCP domain, and the
NADPH-binding site (19), whereas the small one includes the rest of the
protein. The same pattern of degradation was observed upon digestion
with elastase or after long term storage of the protein. The main point
of cleavage (Arg1140-Ile1141) is located just
at the N-terminal end of a region rich in aspartic and glutamic acids
(1150EDDDME1155), which are thought to
provide flexibility together with alanine and proline (37). In the same
way, the C-terminal end of fragments T3, T4, and T8, and hence the
boundary between the PCP and the NADPH-binding domains, is likely to
lie at another putative linker region
(954AANEPDDE961). This result was unexpected
considering the putative individual domains predicted by using sequence
comparisons (15, 19), where the NADPH binding site was believed to be
included within the reductive domain, and suggests that the nucleotide
binding site could belong to an individual domain that under the
proteolytic conditions used in this study is associated to the PCP
domain. Indeed, under harsher conditions this nucleotide-binding box is never found as an independent fragment and is degraded, which suggests
a less structured organization than that of other domains. This
behavior is not unprecedented and has been observed with structural
domains of other multidomain systems (35, 38).
-AAR was performed in the absence of NADPH to prevent
reductive cleavage of a putative acyl-adenylate (5). The
time-dependent accumulation of radioactivity observed (Fig. 3) and its subsequent release upon addition of NADPH are in agreement with
-[3H]AA being presumably activated in the
adenylation domain and then transferred to the PCP domain, where it
forms a transient acyl-thioester intermediate previous to its
hydrolysis by the reductase domain (Fig. 5) (5). The selective release,
of labeled
-AA from the acylated
-AAR by performic acid but not
by formic acid treatment, indicates that the
-AA is bound to the
enzyme through a thioester linkage, supporting the involvement of the phosphopantetheine arm on the transfer of the activated substrate to
the reduction domain (R in Fig. 5).
-aminoadipate reductases and non-ribosomal
peptide synthetases.
-[3H]AA provided evidence that both
domains retain activity after proteolysis and supports the former
model, where the PCP domain would covalently tether the acyl-thioester.
The radioactivity observed in fragment T6 (which lacks the PCP domain)
goes beyond, suggesting that the adenylation domain is catalytically
competent even in the absence of its C-terminal region and supporting
the assumption that such domain functions in the loading of free
-AA (5, 15, 19). Similar PCP-independent non-covalent substrate binding of
an adenylation domain has been reported for the
phenylalanine-activating domain of the gramicidin S synthetase, GrsA
(41).
-[3H]AA or more likely that there is a collaboration
with the adenylation domain in trans. Such in
trans collaboration between functional domains resembles the
mode of action of multienzyme complexes and has also been described for
individual domains of multifunctional enzymes (42). In summary this
work has allowed us to gain insight into how this fascinating enzyme operates.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Prof. P. F. Leadlay (Cambridge, UK) for help with determination of the N-terminal sequences and Dr. J. Casqueiro for the kind gift of the strain P. chrysogenum AMA, and M. Corrales, B. Martín, and J. Merino for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Comisión Interministerial de Ciencia y Tecnología, Madrid, Spain (Grants BIO97-0289-C02-01 and BIO20001726-C02-01) and by Antibióticos, S.p.A., Milán, Italy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Received a fellowship from the Basque Government, Vitoria, Spain.
To whom correspondence should be addressed. Tel.:
34-987-210-308; Fax: 34-987-210-388; E-mail: degjmm@unileon.es.
Published, JBC Papers in Press, December 31, 2002, DOI 10.1074/jbc.M211235200
2 F. Teves, J. Casqueiro, and J. F. Martín, unpublished results.
3 F. J. Casqueiro and J. F. Martín, unpublished data.
4 M. J. Hijarrubia and J. F. Martín, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
-AA,
-aminoadipate;
-AAR,
-aminoadipate reductase;
ACV,
-L-(
-aminoadipyl)-L-cysteinyl-D-valine);
PCP, peptidyl carrier protein;
PBS, phosphate-buffered saline;
MES, 4-morpholineethanesulfonic acid.
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