From the Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Fachgebiet Biochemie und Molekulare Biologie, Technische Universität Berlin, Franklinstrasse 29, D-10587 Berlin-Charlottenburg, Germany
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ABSTRACT |
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Actinomycin synthetase I (ACMS I) activates
4-methyl-3-hydroxyanthranilic acid, the precursor of the chromophoric
moiety of the actinomycin, as adenylate. The gene acmA of
ACMS I was identified upstream of the genes acmB and
acmC encoding the two peptide synthetases ACMS II and ACMS
III, respectively, which assemble the pentapeptide lactone rings of the
antibiotic. Sequence analysis and expression of acmA in
Streptomyces lividans as enzymatically active
hexa-His-fusion confirmed the acmA gene product to be ACMS
I. An open reading frame of 234 base pairs (acmD), which
encodes a 78-amino acid protein with similarity to various acyl carrier
proteins, is located downstream of acmA. The
acmD gene was expressed in Escherichia coli as
hexa-His-fusion protein (Acm acyl carrier protein (AcmACP)). ACMS I in
the presence of ATP acylated the purified AcmACP with radioactive
p-toluic acid, used as substrate in place of 4-MHA. Only
10% of the AcmACP from E. coli was acylated, suggesting
insufficient modification with 4'-phosphopantetheine cofactor.
Incubation of this AcmACP with a holo-ACP synthase and coenzyme A
quantitatively established the holo-form of AcmACP. Enzyme assays in
the presence of ACMS II showed that toluyl-AcmACP directly acylated the
thioester-bound threonine on ACMS II. Thus, AcmACP is a 4-MHA
carrier protein in the peptide chain initiation of actinomycin synthesis.
The actinomycins, a family of bicyclic chromopeptide lactones with
strong antineoplastic activity (1), are produced by various
streptomycete strains (2, 3). Common to all actinomycins is the
chromophoric moiety actinocin, a unique phenoxazinone dicarboxylic acid, to which are attached two pentapeptide lactone rings in amide
linkage (Fig. 1). Biogenetic studies have shown that actinocin is
derived from 4-methyl-3-hydroxyanthranilic acid
(4-MHA),1 a metabolite from
tryptophan (4). However, actinocin is not synthesized from 4-MHA
directly. Instead, phenoxazinone formation takes place by oxidative
condensation of preformed 4-MHA pentapeptide lactones most
probably catalyzed by a phenoxazinone synthase (5) (Fig. 1).
The assembly of the 4-MHA pentapeptide lactones proceeds in a
nonribosomal mechanism (5, 6). Two peptide synthetases of 280 and 480 kDa (ACMS II and ACMS III, respectively) were previously identified in
actinomycin-producing Streptomyces chrysomallus carrying the
five amino acid modules responsible for assembly of the pentapeptide
lactone rings as indicated in Fig. 1 (7, 8). ACMS II and III were shown to contain also the functions for
epimerization of the valine in position 2 and for introduction of the
methyl groups into sarcosine and N-methylvaline in positions 4 and 5, respectively (7-9).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structure of actinomycin and 4-MHA
pentapeptide lactone and scheme of their assembly by the
actinomycin synthetases. Actinomycin is formed by oxidative
condensation of two 4-MHA pentapeptide lactones. The 4-MHA pentapeptide
lactone is assembled on actinomycin synthetases II and III, which
activate the five amino acids of the peptide lactone ring in the
indicated order. 4-MHA is adenylated by ACMS I. Peptide synthesis
starts after acylation of threonine with activated 4-MHA on the surface
of ACMS II. Sar, N-methylglycine; MeVal,
N-methyl-L-valine; AdoMet,
S-adenosyl-L-methionine.
4-MHA, the ultimate amino-terminal residue of the 4-MHA pentapeptide lactone sequence, is activated by a separate adenylating enzyme, ACMS I (10). It has a size of 45 kDa and can adenylate a variety of benzene carboxylic acids structurally related to 4-MHA, e.g. 4-methyl-3-hydroxybenzoic acid (4-MHB) or p-toluic acid (10, 11). These compounds can replace 4-MHA in the 4-MHA pentapeptide lactone when fed to actinomycin-producing Streptomyces through competition with the endogenous 4-MHA, giving rise to new acyl pentapeptide lactones (12). ACMS I does not detectably bind its substrate as thioester, and therefore the corresponding binding domain would be located on an acceptor protein distinct from ACMS I. Incubation of peptide synthetase ACMS II purified from S. chrysomallus with threonine and p-toluyl-CoA-thioester or p-toluyl-pantetheine-thioester, both nonnatural substrates mimicking a 4'-phosphopantetheine thioester, resulted in the formation of covalently bound p-toluyl-threonine (8). ACMS II also catalyzed, albeit with lower efficiency than in the latter cases, formation of p-toluyl-threonine from threonine and chemically synthesized p-toluyl-adenylate. This suggested that ACMS II, which cannot activate 4-MHA, would possess a binding domain with a 4'-phosphopantetheine cofactor (ACP domain) as an acceptor site for 4-MHA (8).
Cloning of the genes of ACMS II and III (acmB and
acmC) showed that they lie closely linked in tandem on the
chromosome of S. chrysomallus (13). Their analysis revealed
an organization of the ACMSs into two and three modules, respectively,
necessary for pentapeptide lactone assembly. Inspection of the protein
sequence of ACMS II, however, showed that the conserved motif of the
4'-phosphopantetheine cofactor attachment site occurred only twice,
namely in the two amino acid modules of this protein. Thus, the third
4'-phosphopantetheine cofactor, which was initially postulated to lie
in front of the first (threonine) module (8), is not located on ACMS
II. From the previous work on acmB and acmC in
the acm gene cluster we got evidence that the 5'-end of the
gene acmA of ACMS I is located upstream of the gene
acmB of ACMS II and points in the opposite direction (13).
Here we show the cloning and expression of acmA and the
analysis of the gene product. More importantly, we found directly
downstream of the gene of ACMS I a small open reading frame (ORF)
encoding a small ACP (acmD), which is the protein harboring
the missing third 4'-phosphopantetheine cofactor required for
condensation of 4-MHA with threonine on ACMS II.
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EXPERIMENTAL PROCEDURES |
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Chemicals and Radiochemicals--
4-MHA was synthesized as
described (10). p-[1-14C]Toluic acid (8.3 Ci/mol) was from Sigma. Tetrasodium-[32P]pyrophosphate
(28.2 Ci/mmol) was from NEN Life Science Products, and
[-32P]ATP (400 Ci/mmol) was from Hartmann Analytik
(Braunschweig, Germany). All other chemicals were of the highest grade
commercially available.
Strains and Cultures--
Streptomyces lividans 1326 was from the John Innes strain collection (Norwich, United Kingdom). It
was grown and kept on R2YE (14). Submerged growth was for up to 4 days
at 30 °C with shaking (250 rpm) in 300-ml Erlenmeyer flasks
containing 100 ml of YEME medium (14). Flasks were equipped with steel
springs as baffles. Escherichia coli strains for cloning
were DH5 (15) and JM109 (16), and the strain for expression was M15,
from Qiagen. They were grown according to standard protocols.
Plasmids, DNA Manipulations, and Cloning and Sequencing Procedures-- Techniques for DNA isolation, manipulation, and transformation were as described by Sambrook et al. (17) and Hopwood et al. (14). The cosmid cosA1 carrying the ACMS gene cluster from S. chrysomallus ATCC 11523 was described previously (13). The plasmids used for subcloning fragments were pTZ18U (Amersham Pharmacia Biotech), pSP72 (Promega), pSL1180 (Amersham Pharmacia Biotech). Sequence determinations were performed with a Taq cycle sequencing kit (United States Biochemicals-Amersham Pharmacia Biotech sequencing kit US71001 or US78500) on plasmid DNA. Fragments were subcloned into pTZ18U. Sequence comparisons, multiple sequence alignments, and identity scores were computed with CLUSTAL W (18) or with the FASTA data base search results (19). The plasmid for expression of acmA and acmB in S. lividans was pIJ702 (20). For expression of acmA in E. coli, expression vector pQE30 (Qiagen) was used, and for expression of acmD in E. coli, pQE32 (Qiagen) was used. For PCRs, Vent DNA polymerase (BioLabs) was used according to the manufacturer's instructions.
Heterologous Expression of acmA-- Expression of acmA in E. coli was as amino-terminal hexa-His-tagged fusion protein. The acmA was amplified with suitable restriction ends by PCR using cosmid cosA1 (13) as template. Forward and reverse primers were FACMA1 (5'-TAAGAGGAAGCTGGATCCGCCGATAAATGGTG-3') and RACMA2 (5'-TAGGCGTGGATCCCGTCGACCGAGGTGAA-3'), respectively. The resulting 1.6 kilobase pair PCR fragment was digested with BamHI and SalI and ligated into pQE30. In this construct the amino-terminal end of ACMS I would change from MADK- to MRGSHHHHHHGSADK-. Transformation into E. coli JM109 yielded plasmid pACMA1. After restriction analysis and control sequencing on both ends of the fragment, pACMA1 was transformed into E. coli strain M15. Cultures of M15/pACMA1 (1.6 liters of 2× YT medium, 100 µg/ml ampicillin, 25 µg/ml kanamycin) were grown at 30 °C to an A600 of 0.9 and then induced with 2 mM isopropylthiogalactoside. Cells were harvested after a further 14 h of incubation at 30 °C.
For the expression of acmA as carboxyl-terminal
hexa-His-tagged fusion protein in S. lividans, acmA was
placed as a translational fusion in frame into the ATG start codon of
the melC1 gene of Streptomyces plasmid pIJ702
(20), which is under control of the mel promoter. The ATG
start codon of melC1 is contained in the unique
SphI restriction site of the plasmid. The acmA
was amplified with synthetic primers to generate a matching
5'-SphI site in its start codon and to create a hexa-His
encoding sequence between the last codon and the stop codon. PCR was
performed with primers FACEX2
(5'-GAGGGCATGCATATGGCCGATAAATGGTGGGGGGAA-3') and RACEX2
(5'-GAAGATCTTCAGTGGTGGTGGTGATGGTGCGAGGCCCCCTTGAGCTCAGCGGG-3') using subclone pA1sub27 containing a 2.8-kilobase SalI
fragment from cosA1 cloned into pTZ18U (Fig.
2). A 1.45-kilobase fragment was
obtained, which, after cleavage with SphI and
BglII, was ligated in pSP72 cleaved with SphI and
BglII. After transformation in DH5, plasmid pACMA10 was
obtained. Sequencing on both ends of the insert confirmed the
correctness of the modified ends of acmA. pACMA10 was
digested with SphI and BglII, and the excised
1.45-kilobase SphI/BglII fragment was isolated
and ligated into SphI/BglII-cleaved pIJ702. Transformation into S. lividans resulted in plasmid
pACMA11. The recombinant protein encoded by pACMA11 is changed at the
amino terminus from MADK- to MHMADK- and at the carboxyl terminus from -AS to -ASHHHHHH. Microsequencing of the purified protein later confirmed the new amino-terminal sequence (not shown). For expression of acmA in S. lividans encoded by pACMA11 as
hexa-His-tagged fusion protein, the transformed strain was grown at
30 °C (6 liters of YEME medium, 10 µg/ml thiostrepton) for 4 days
and then harvested by suction filtration.
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Heterologous Expression of acmB-- The acmB was expressed in S. lividans from plasmid pACM5 as described previously (13).
Heterologous expression of acmD (AcmACP)-- The gene acmD encoding the 4-MHA carrier protein was expressed in E. coli as amino-terminal hexa-His-tagged fusion protein. The acmD gene was engineered by PCR with synthetic primers FACPI1 (5'-CCGCATGCTCTCGAAGGACGACATCAGGGCGAT-3') and RACPI1 (5'-CGAGATCTGTCGTCGGGGCGGTCGCGGCGC-3') using pA1sub27 as template. This generated a 276-base pair fragment with a SphI site (5') and the BglII site (3') necessary for ligation into plasmid pTZ18U cleaved with SphI and BamHI. After transformation into E. coli, the resultant plasmid was cleaved with SphI and KpnI, and the excised approximately 250-base pair fragment was ligated into expression vector pQE32 cleaved with SphI/KpnI. Successive transformation into JM109 and in M15 yielded plasmid pACPI-Q1. Sequence analysis of the cloned fragment in pACPI-Q1 confirmed that in the encoded protein, the amino terminus is changed from MISK- to MRGSHHHHHHGIMLSK-. Cultures of M15/pACPI-Q1 (1.8 liters of 2× YT medium, 100 µg/ml ampicillin, 25 µg/ml kanamycin) were grown at 30 °C to an A600 of 0.7 and then induced with 0.1 mM isopropylthiogalactoside. Cells were harvested after further growth for 14 h at 30 °C.
Heterologous Expression of a Putative 4'-Phosphopantetheine Transferase Gene (ORF C, 761 base pairs) flanking the nosiheptide resistance gene of Streptomyces actuosus (21)-- The gene was amplified py PCR with oligonucleotides A (5'-AGCGCGGAATCGACTGAGGATCCATGACGGCCCGACA-3') and B (5'-TGTTGCTCTCAAGCTTGGTCAGATCACGA-3'). Template for PCR was a genomic 8.5-kilobase BamHI fragment in clone 202, kindly provided by H. G. Floss and colleagues. The PCR-generated fragment was digested with BamHI and HindIII at the sites introduced by the oligonucleotides A and B (underlined) and inserted in the E. coli expression plasmid pQE30 (Qiagen), resulting in plasmid pPAN-5. Expression of ORF C from pPAN-5 was performed as described for acmD.
Protein Purification-- All operations were carried out at 0-4 °C.
Purification of Recombinant ACMS I from E. coli M15 Carrying pACMA1-- A suspension of 16 g (wet weight) of cells in 60 ml of cold Buffer AN (10% glycerol (w/v), 50 mM KPO4 buffer (pH 7.0), 1 mM benzamidine, 1 mM PMSF was passed through a French press at 10,000 psi. After treatment with DNase I (Sigma) (20 µg/ml) and stirring for 45 min, the suspension was centrifuged for 10 min at 12,000 × g. The supernatant (65 ml) was passed through a DEAE-cellulose column (9 × 3.5 cm) to which, at pH 7, ACMS I does not bind. Fractions containing enzyme activity were pooled (80 ml). After adding 5 ml of 1 M Tris-HCl (pH 8.0) and 15 ml of Buffer NI (15% glycerol (w/v), 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM PMSF, 1 mM benzamidine), the protein solution was applied to a nickel-chelate column (3.4 × 1.5 cm) equilibrated previously with Buffer NI. After washing with Buffer NW (same as Buffer NI but at pH 7.0 instead pH 8.0), protein bound was eluted with increasing concentrations of imidazole in Buffer NW (40 mM, 145 mM, 250 mM). Fractions with enzymatic activity were pooled (9 ml) and desalted on an AcA202 (Ultrogel) column (12 × 5 cm) equilibrated with Buffer M (10% glycerol (w/v), 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM DTE, 1 mM benzamidine, 1 mM PMSF). Enzyme was purified further by ion exchange chromatography on a MonoQ HR 5/5 column (Amersham Pharmacia Biotech) in Buffer M. Enzyme was eluted with a 40 ml gradient (1 ml/min flow rate) from 0-0.2 M NaCl.
Purification of Recombinant ACMS I Encoded by pACMA11 from
S. lividans--
About 35 g of freshly harvested mycelium of
S. lividans carrying pACMA11 suspended in 70 ml of Buffer A
(10% glycerol (w/v), 50 mM KPO4 buffer (pH
7.0), 1 mM EDTA, 4 mM DTE, 1 mM
PMSF, 1 mM benzamidine) was passed twice through a French
press at 10,000 psi. After DNase I treatment (30 µg/ml) and with
gentle stirring for 90 min, the suspension was centrifuged (30 min at
25,000 × g). The supernatant (60 ml) was passed
through a DEAE-cellulose column (8 × 3.5 cm) to which ACMS I does
not bind at neutral pH. Fractions with enzymatic activity were pooled
(240 ml). Addition of 28 ml of 1 M Tris-HCl, pH 8.0, shifted pH from 7.0 to 8.0, and the solution was applied to a
nickel-chelate resin column (2.8 × 1.6 cm). After washing the
column with Buffer NW (see above), bound proteins were eluted with a
150-ml linear gradient of 0-250 mM imidazole in Buffer NW.
Fractions of 3.5 ml were collected, and the fractions with enzymatic
activity was pooled. This pool was diluted with 5 volumes of 15%
glycerol (w/v), 4 mM DTE, 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine and applied onto an
-aminohexyl-Sepharose 4B (Sigma) column (6.5 × 1 cm) for
enzyme concentration. After washing with Buffer C (15% glycerol (w/v),
50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4 mM DTE, 1 mM PMSF, 1 mM
benzamidine), the bound protein was eluted with a step of 0.3 M NaCl in Buffer C. The protein (total volume, 6 ml) was
purified in 1.5-ml portions to homogeneity by gel filtration on an
SuperdexTM75 column (Amersham Pharmacia Biotech) previously
equilibrated with Buffer B (15% glycerol (w/v), 100 mM
Tris-HCl (pH 8.0), 1 mM EDTA, 4 mM DTE, 1 mM PMSF, 1 mM benzamidine). Protein with apparent homogeneity was in the rear half of the activity peak. The
purified recombinant protein could be stored at
80 °C for at least
6 months without detectable loss of activity.
Purification of 4-MHA Carrier Protein (AcmACP) Expressed in E. coli--
Approximately 12 g of cell paste was suspended in 34 ml
of Buffer NI (see above). After passage of the suspension through a
French press at 10,000 psi, DNase I (25 µg/ml) was added, and the
suspension was left on ice for 15 min with gentle stirring. After
centrifugation (15 min at 20,000 × g), the supernatant
was applied to a nickel-chelate column (3 × 1.6 cm) equilibrated
with Buffer NI. The column was washed with 15 ml of the same buffer and
80 ml of Buffer NW until no more protein eluted from the column. A
100-ml linear gradient of 0-500 mM imidazole in Buffer NW
afforded elution of hexa-His-tagged protein. The bulk of protein
appeared at 100-200 mM imidazole, and the corresponding
fractions were pooled. 1 M DTE was added to give 4 mM final concentration, and 4.5-ml portions (3 mg protein)
were gel filtrated on Ultrogel AcA54 (40 × 25 cm column
dimensions) in Buffer B (see above). After this step, enzyme appeared
to be pure as judged from SDS-PAGE. Typically, about 4 mg of total
protein were obtained by this procedure. Protein could be stored at
80 °C for 4 weeks without loss of p-toluic binding
activity in Buffer B.
Purification of Recombinant ACMS II Encoded by pACM5 from S. lividans-- Partially purified recombinant ACMS II was prepared as described previously (13). Enzyme from the AcA34 gel filtration step was used.
Purification of Recombinant 4'-Phosphopantetheine Transferase (Holo-ACP Synthase) from S. actuosus Encoded by pPAN5-- The resultant hexa-His-tagged fusion protein was purified analogous to AcmACP as described above.
Analytical Methods-- SDS-PAGE was performed according to Laemmli (22). Protein determinations were done according to Bradford (23). Antibodies against ACMS I from S. chrysomallus were raised in a rabbit by two administrations of 100-µg portions of the purified enzyme (at a 1-month interval) (courtesy of Prof. F. J. Fehrenbach, Robert-Koch-Institut, Berlin, Germany). The serum was used without further purification in dilutions of 1:1000 to 1:3000 for Western blot analysis by semidry blot standard techniques (provided by Biometra and Schleicher & Schuell). Anti-rabbit-alkaline phosphatase conjugate (Sigma) served as second antibody for the purpose of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate detection (both from Roche Molecular Biochemicals). Thin-layer chromatography of enzymatically formed acyl adenylates was performed as described (10).
Enzyme Assays--
ATP-[32P]pyrophosphate exchange
reactions and acyl adenylate formation tests with
[-32P]ATP were as described previously (10, 11).
The recombinant 4-MHA carrier protein (AcmACP) was tested for thioester formation with p-[1-14C]toluic acid (used as 4-MHA substrate analogue) by measuring the formation of trichloroacetic acid-precipitable radioactivity. One unit of AcmACP was defined as the amount of enzyme that covalently binds 1 nmol of p-toluic acid in 30 min at 29 °C. Standard assay contained 14 mM p-[1-14C]toluic acid, 10 mM ATP, 45 mM MgCl2, 280 nM purified recombinant ACMS I and 100 µl of AcmACP-containing protein fraction. Buffer was Buffer B (see above) and incubation was at 29 °C for 30 min. After addition of 6% trichloroacetic acid and leaving on ice for 15 min, precipitated proteins were collected by suction filtration on membrane filters ME 25 (Schleicher & Schuell). After washing and drying, filter bound radioactivity was counted in a Packard 1600CA scintillation counter. To visualize charged AcmACP as labeled band in SDS-polyacrylamide gels, precipitated protein was resuspended in 70 µl of of 15% (w/v) glycerol, 100 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM PMSF, and 1% SDS. 2 µl of 40% sucrose-0.25% bromphenol blue was added. 5-µl aliquots were usually counted by liquid scintillation counting, and 10-µl portions were separated by SDS-PAGE (17.5% polyacrylamide). Gels were stained with Coomassie Brilliant Blue R250 (Serva) and, after drying, exposed to x-ray film (NIF100, Konica).
Measurement of activity of recombinant ACMS II was as described previously (13).
The assay to demonstrate the transfer of p-toluic acid from p-toluic acid-AcmACP-thioester to ACMS II-threonine thioester was the same as described above to measure charging of AcmACP but with the additional presence of ACMS II and its substrate threonine. Assays contained 200 nM ACMS I, 5.1 µM AcmACP (0.35 units), 0.046 units of partially purified ACMS II, 10 mM ATP, 50 mM MgCl2, 14 mM p-[1-14C]toluic acid, 8 mM threonine and were incubated for 30 min at 29 °C. After addition of 5% trichloroacetic acid, protein was allowed to precipitate overnight on ice. Control reactions involved omission of one component from the reaction in each case. Precipitated proteins were collected, washed twice with 7% trichloroacetic acid and once with 6 ml of 3% trichloroacetic acid, and finally resuspended in a 350-µl solution of 15% (w/v) glycerol, 100 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM PMSF, and 1% SDS. 9 µl of 40% sucrose-0.25% bromphenol blue was added. A 10-µl aliquot was analyzed by liquid scintillation counting, and 8 µl was applied onto a 5% SDS-polyacrylamide gel. Staining, drying, and autoradiography was as above.
Incubation of AcmACP with the holo-ACP synthase from S. actuosus was performed in a total volume of 600 µl (Buffer B) at 30 °C for 45 min in the presence of 28 µM AcmACP, 1 µM holo-ACP synthase, 290 nM ACMS I, 10 mM p-[1-14C]toluic acid, 10 mM ATP, 20 mM MgCl2 and 0.2 mM CoA. 80-µl aliquots were precipitated with 2 ml of 7% trichloroacetic acid and 50 µl of 3% bovine serum albumin at several time points. The trichloroacetic acid-precipitable and filter bound radioactivity was measured as described above (filter GF92, Schleicher & Schuell).
Nucleotide Sequence Accession Number--
The nucleotide
sequences from S. chrysomallus obtained in this study has
been assigned GenBankTM accession number AF134587 for
acmA and AF134588 for acmD.
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RESULTS |
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Cloning and Analysis of the ACMS I Gene-- The genes of the actinomycin synthetases II and III, acmB and acmC, are tandemly arranged on the S. chrysomallus chromosome (13). Analysis of the region upstream of acmB revealed, at a distance of 430 base pairs of noncoding DNA, the start of an ORF in opposite direction to acmB (Fig. 2). The 5'-end of this ORF encodes an amino acid sequence that is identical to the amino-terminal protein sequence of ACMS I, previously determined by microsequencing of the protein (13). From this it became evident that this ORF is the gene coding for ACMS I (acmA). Sequencing further into the 3'-direction revealed the complete ORF of a total length of 1419 base pairs, which would encode a protein of 472 amino acids, with a calculated molecular mass of 51.5 kDa and a pI of 7.36. This fits with the previous determined molecular mass of native ACMS I (52-53 kDa (10)) but is 6.5 kDa larger than the size of denaturated ACMS I estimated from SDS-PAGE (45 kDa) (11). The codon usage of acmA is typical for streptomycete genes showing a strong bias for G+C-rich codons with a G+C-contents of 90.4% at the third base in codons. The overall G+C content of the gene is 71.4%.
Analysis of the deduced protein sequence revealed similarity (between 20 and 30% identity) with a number of aromatic and heteroaromatic carboxylic acid adenylating enzymes, acyl-CoA ligases and the activation domains of various peptide synthetases (Table I). Among these are the aromatic carboxylic acid-activating enzymes EntE and YbtE from E. coli and Yersinia pestis, respectively (24-26). Furthermore, the enzyme has sequence similarity (20% identity) to the hydroxypicolinic acid-adenylating enzyme SnbA from Streptomyces pristinaespiralis and to coumarate CoA ligase (24% identity) from Petroselinum crispum, a member of the acyl-CoA ligases (27, 28). The conservation in these sequences was always highest in the five so-called core regions characteristic for adenylating domains (29) (Table I), which leaves no doubt about the nature of the cloned gene. As predicted from the biochemical analyses of ACMS I obtained from S. chrysomallus (10, 11), no 4'-phosphopantetheine attachment site was found in the enzyme sequence that distinguishes this enzyme from typical peptide synthetases, which bind their substrates as thioesters after activation as adenylate.
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Expression of acmA-- To further characterize the acmA gene product, the gene was expressed in E. coli as an amino-terminal hexa-His-tagged fusion protein. Western blotting of protein extracts of E. coli M15 carrying the expression construct pACMA1 (see under "Experimental Procedures") with antibodies directed against ACMS I from S. chrysomallus showed the presence of the expressed protein as a 45-kDa band in SDS-PAGE (not shown). Nickel-chelate chromatography of total extract from 6 M guanidine hydrochloride-treated cells and in parallel to the soluble fraction of nondenatured cells revealed that by far, most of the protein was in the insoluble fraction. Testing the soluble fraction revealed enzymatic activity as judged from the ATP-pyrophosphate exchange dependent on 4-MHA, 4-MHB, or p-toluic acid at an appreciable activity. The enzyme was purified with reference to the purification of the original protein from S. chrysomallus (see under "Experimental Procedures"). However, this resulted in very low enzyme yields (final total yield, 0.2% at a specific activity of 1.6 nanokatal/mg of protein), which made production of the enzyme in E. coli not amenable for our purposes.
To address the production of active enzyme in higher yields,
acmA was engineered as a translational fusion into the ATG
start codon of the melC1 gene on plasmid pIJ702, and the
resultant plasmid, pACMA11, was transformed into S. lividans. In contrast to the construct used in E. coli,
here the enzyme carried the hexa-His tail at its carboxyl-terminal end.
Analyzing total and cytosolic protein extracts from the transformed
strain and the control strain harboring pIJ702 by Western blotting
revealed the presence of ACMS I after expression of acmA in
S. lividans (Fig. 3).
Moreover, the protein was exclusively in the soluble fraction (not
shown). Total activity based on the protein present in the starting
material was measured by the 4-MHB dependent ATP-pyrophosphate exchange and was found to be 3-fold higher than total enzyme activity in extracts of S. chrysomallus X2-18, an actinomycin-high
producer (11). The purification is shown in Table
II. Remarkably, about 50% of total
activity did not bind to nickel-chelate matrix due to either masking of
the hexa-His tail or its removal through proteolytic activities.
Nevertheless, steps following such as adsorption to
aminohexyl-Sepharose with subsequent elution with a salt step and gel
filtration on SuperdexTM 75 afforded purification of the
enzyme to homogeneity with 12% yield and a specific activity of
approximately 20 nanokatal/mg of protein. This specific activity is
about 3-fold higher than estimated for the wild type enzyme, which
apparently is due to the much shorter purification procedure of the
recombinant protein (four versus eight steps). The
recombinant enzyme was indistinguishable from the wild type enzyme with
respect to acyl adenylate formation both from various benzene
carboxylic acids and in the ATP-pyrophosphate exchange reaction
dependent on these substrates (11). Likewise, the wild type enzyme, the
recombinant ACMS I, did not catalyze the formation of a CoA thioester
from any of the benzene carboxylic acids tested including 4-MHA.
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Cloning and Sequencing of acmD, an ORF Transcriptionally Coupled to acmA-- In the course of sequencing the ACMS I gene acmA, immediately downstream of acmA, an ORF was identified that overlaps with its ATG start the stop codon of acmA indicating transcriptional and translational coupling between the two genes (Fig. 2). This suggested a close functional link between the gene products of acmA and that ORF, which was named acmD. The acmD gene has a length of 236 base pairs, encoding a protein of 78 amino acids in length with a calculated molecular mass of 8691 Da and a pI of 3.83. Comparison of the deduced amino acid sequence of acmD with protein sequences in the data bank revealed similarity with various ACPs from bacteria involved in fatty acid and polyketide synthesis (Table III). These carry 4'-phosphopantetheine as prosthetic group attached to a conserved serine, which is also present in the acmD gene product (Table III). There was also similarity with DltC, the D-alanine carrier in lipoteichoic biosynthesis in Lactobacillus casei (30), the ACP domain of the entB gene product of the enterobactin synthesis system in E. coli (31, 32), and the amino-terminal ACP extradomain of the yersiniabactin synthetase HWMP2, a peptide synthetase involved in the biosynthesis of the acyltripeptide yersiniabactin in Yersinia strains (33, 34). The gene product of acmD appeared to be a suitable candidate for carrying the missing 4'-phosphopantetheine cofactor accepting 4-MHA in actinomycin biosynthesis and was therefore named AcmACP.
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Expression of acmD as a Hexa-His-tagged Fusion Protein--
For
expression of acmD as hexa-His-tagged fusion protein the
expression plasmid pACPI-Q1 (using vector pQE 32) was constructed. After transformation of E. coli strain M15 cultivation was
performed at 30 °C and induction was with low concentrations of
isopropylthiogalactoside to allow gradual expression of the gene.
SDS-PAGE analysis of protein extracts of the E. coli strain
transformed with plasmid pACPI-Q1 revealed an abundant protein of
approximately 10 kDa, which was missing in the control strain carrying
pQE32 (Fig. 4, lanes 1 and
2, respectively). The purification of the protein was by
nickel-chelate chromatography and Ultrogel AcA 54 gel filtration, which
yielded an appreciable amount of pure protein (Fig. 4, lane 3). The total yield was 67%.
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The ability of the protein to bind
p-[14C]toluic acid in the presence of ACMS I
and ATP was measured by the formation of trichloroacetic acid-stable
radioactivity. Covalent substrate binding was also used to demonstrate
the charged protein as a labeled band in SDS-PAGE as described under
"Experimental Procedures." Fig. 4, lanes 4 and
4', shows that the protein was charged with
p-[14C]toluic acid when ACMS I and ATP were
present. No radiolabeling of the protein was seen when ATP (Fig. 4,
lanes 5 and 5') or ACMS I (Fig. 4, lanes
3 and 3') were absent in the reaction mixture. These
findings indicate that the AcmACP does not activate p-toluic acid per se and was charged only in case of prior activation
of p-toluic acid by ACMS I. An estimate for the extent of
4'-phosphopantetheinylation was obtained by comparing the amount of
radioactive substrate covalently bound to the protein and the total
amount of AcmACP protein present in the assays. In two AcmACP
preparations after gene expression in E. coli, only 10% of
the AcmACP was found to bind ACMS I activated p-toluic acid.
This apparently points to a low extent of phosphopantetheinylation of
AcmACP in the foreign host E. coli. To increase the amount
of phosphopantetheinylated AcmACP (holo-AcmACP) and also to demonstrate
that the p-toluic acid became bound to AcmACP via
4'-phosphopantetheine as prosthetic group, the acylation of AcmACP by
ACMS I was performed in the additional presence of a holo-ACP synthase
and CoA (35). We chose for these experiments the holo-ACP synthase from
S. actuosus (21), which was expressed from its gene as a
hexa-His-tagged fusion protein in E. coli as described under
"Experimental Procedures." The holo-ACP synthase was added to the
AcmACP/ACMS I incubations in 1 µM concentrations and
incubated for different lengths of time with and without CoA. The data
in Fig. 5 clearly show that with
increasing length of incubation, the amount of p-toluic acid covalently bound to AcmACP increased until the final level of 95-100%
mol of p-toluic acid/mol of AcmACP was reached. By contrast, in the absence of CoA the basal level of 10% remained constant during
the whole 40 min of incubation, which was also the case when no
holo-ACP synthase was present (the latter not shown). These data
illustrate the 1:1:1 stoichiometry between the enzyme, its cofactor,
and the substrate p-toluic acid. Moreover, varying the ACMS
I concentration (from 20 to 450 nM) in short (2 and 4 min)
incubations with AcmACP revealed direct proportionality between the
amount of ACMS I added and p-toluyl-AcmACP formed (not
shown).
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p-Toluyl-AcmACP Is Substrate of ACMS II--
Once we had
identified the acmD gene product as 4-MHA binding protein,
we set out to elucidate the possible interaction between the AcmACP and
ACMS II in the transfer of the aromatic carboxylic acid to the
threonine moiety (i.e. the first amino acid in the pentapeptide lactone ring) activated by ACMS II. In order to exclude possible contamination of ACMS II with AcmACP when isolated from actinomycin-producing S. chrysomallus, we used the
recombinant ACMS II expressed in S. lividans (13). This
ensured that all components used for the enzyme reaction were derived
from an actinomycin-free background. Data in Fig.
6 (lanes 4 and 4')
show that upon incubation of ACMS II with AcmACP, ACMS I and their
substrates threonine, p-[14C]toluic acid, and
ATP, the bands of both ACMS II and AcmACP were significantly labeled.
When the same experiment was performed without ATP (Fig. 6, lanes
3 and 3') or without the AcmACP (lanes 5 and
5'), labeling of neither protein band was observed. This indicates that binding of p-toluic acid is
ATP-dependent and that labeling of ACMS II only occurs from
the charged AcmACP. In the absence of threonine, ACMS II became only
faintly labeled, possibly due to the presence of residual trace amounts
of threonine that had been loaded to the enzyme previously in in
vivo conditions. (Fig. 6, lanes 2 and 2')
This findings clearly show that the p-toluic acid bound to
AcmACP directly acylates the threonine covalently bound to ACMS II.
Lanes 6 and 6' in Fig. 6 show that when ACMS I is
absent, neither band of AcmACP nor ACMS II are labeled, which clearly
reveals that charging of both AcmACP and ACMS II is dependent on the
prior activation of p-toluic acid as adenylate.
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DISCUSSION |
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We have shown previously that the 4-MHA pentapeptide lactones, the penultimate precursors of the bicyclic actinomycins, are assembled by the two peptide synthetases, ACMS II and III. These enzymes from actinomycin-producing S. chrysomallus activate the five amino acids of the peptide lactone ring as adenylates and thioesters, whereas 4-MHA is activated by a separate 4-MHA adenylating enzyme, ACMS I. ACMS I does not bind its substrate as thioester as revealed by enzymatic testing and, as shown here, because of the absence of a 4'-phosphopantetheine attachment site in its sequence. Furthermore, ACMS I has no activity as an acyl-CoA ligase (11). It was shown previously that purified ACMS II binds threonine, the first amino acid of the pentapeptide chain, as thioester, which can be acylated with nonnatural substrates such as p-toluyl-CoA or p-toluyl-phosphopantetheine thioesters, yielding p-toluyl-threonine (8). This suggested that 4-MHA (represented in these experiments by the structural analogue p-toluic acid) in natural conditions would be thioesterified to an unknown carrier, which most probably contained a 4'-phosphopantetheine cofactor (8). Moreover, purified ACMS II from S. chrysomallus reacted, when charged with threonine, with chemically synthesized p-toluic acid-adenylate under formation of p-toluyl-threonine, suggesting that the 4'-phophopantetheine cofactor would be located on ACMS II. This view was supported by the fact that covalent binding of p-toluic acid to enzyme could be inhibited by sulfhydryl blockers, such as dibromopropanone, n-ethylmaleinimide, or iodoacetamide (8). Sequencing the ACMS II gene, however, revealed that ACMS II had only two 4'-phosphopantetheine attachment sites, each located in the ACP domain of the threonine and valine modules (13).
The data presented here clearly show that the postulated
additional 4'-phosphopantetheine cofactor is located on a small 4-MHA carrier protein (AcmACP) encoded by acmD located downstream
of the ACMS I gene acmA in the ACMS gene cluster (Fig. 2).
The functional studies done with the purified protein expressed in the
foreign host E. coli clearly reveal that AcmACP is
specifically acylated by ACMS I with p-toluic acid (or
4-MHA). The acylated AcmACP then is used as substrate by ACMS II in the
acylation of the covalently bound threonine. This is most probably with
the assistance of the putative acyltransferase domain containing the
HHIVMDAFG motif in the amino-terminal region of ACMS II found in front
of the threonine module (Fig. 7). These
results clearly assign the role as carrier in the transfer and
thioesterification of 4-MHA to the acmD gene product and not
to an ACP extradomain located on ACMS II.
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The involvement of small ACPs and of ACP extradomains in peptide
biosynthesis systems or related systems in bacteria has been realized
only recently. A first example is the 8.9-kDa D-alanine carrier protein
from Lactobacillus casei, which carries D-alanine through
the cell membrane to membrane-associated lipoteichoic acid and, in
cooperation with an acceptor, forms a D-alanine ester with lipoteichoic
acid (30, 36). In the biosynthesis of the trilactone, enterobactin,
which is a trimer of 2,3-dihydroxybenzoyl-serine (2,3-DHB-serine), is
assembled by EntE, a dihydroxybezoic acid adenylating enzyme, and EntF,
a serine-activating peptide synthetase (25, 37). Their roles are
analogous to those of ACMS I and ACMS II in the assembly of
4-MHA-threonine (reviewed in Ref. 6). Recent work has revealed that
EntB, the isochorismate lyase that catalyzes the last step of 2,3-DHB
synthesis carries an ACP extradomain of 100 amino acids in length (32).
This ACP domain was identified as binding domain for 2,3-DHB, which
could be modified with 4'-phosphopantetheine cofactor in
vitro by the holo-ACP synthase EntD (32). EntB was subsequently
shown to be an essential compound in the synthesis of the whole
enterobactin in a reconstituted cell-free system (33). The role of EntB
thus is analogous to the role of AcmACP described here. Interestingly,
the iron-chelating acyl tripeptide yersiniabactin contains salicylic
acid at its amino-terminal end. Sequence analyses of the yersiniabactin
gene cluster in Yersinia pestis (26) showed a gene encoding
YbtE, the salicylic acid-activating enzyme, and two genes encoding the
peptide synthetases, HMPW1 and HMPW2, responsible for yersiniabactin
peptide assembly. HMPW2 activates cysteine, the first amino acid in the
yersiniabactin biosynthesis sequence (38), and was shown to be the
partner of YbtE in a fashion similar to EntE and EntF. Remarkably, the salicylyl-ACP domain in the yersiniabactin system is fused to the amino
terminus of HMPW2, which transfers salicylic acid to the covalently
bound cysteine with formation of salicylyl-cysteine. The same domain
organization as in HMPW2 with respect to the ACP domain had initially
been postulated by our group for ACMS II based on the biochemical
analysis of this enzyme prepared from S. chrysomallus (8).
However, 4-MHA peptide synthesis differs from these examples in that
AcmACP is a separate small acyl carrier protein and not fused to any
protein of the same pathway. Fusion of ACPs to other proteins of the
same pathway may ensure their coexpression. In the case of actinomycin
biosynthesis, fusion of the AcmACP to another enzyme of the pathway or
an ACMS did not happen in S. chrysomallus. Apparently,
cotranscription and cotranslation of acmA and
acmD suffices to ensure the coordinate expression with each
other and the other actinomycin biosynthesis genes. The separate acyl
carrier proteins or extradomains in the biosynthesis systems of
aromatic acylpeptides and peptide lactones represent the functional
link between the adenylating enzymes and the large modular peptide
synthetases, which consist of similar repeating units (modules)
activating amino acids only. ACPs or ACP domains carrying the aromatic
carboxylic acids can specifically acylate the activated substrates
residing on a peptide synthetase. Remarkably, CoA is not used as
carrier. This appears also to be the case in the formation of several
macrocyclic polyketides, which contain aromatic or pseudoaromatic
carboxylic starter units, such as shikimic acid or
3-amino-5-hydroxybenzoic acid in the biosynthesis of rapamycin and
FK506 (39, 40) or rifamycin (41), respectively. The corresponding
polyketide synthases contain starter modules with a domain with
similarity to acyl-CoA ligases and, in addition, an ACP domain. It may
be concluded that the ACP domain is directly acylated by the CoA ligase
domains without intermediation of the aryl CoA derivative. Formation of
CoA thioesters of aromatic carboxylic acids in bacteria thus may be a
route only in the catabolism of aromatic compounds.
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ACKNOWLEDGEMENTS |
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We thank Prof. Dr. F. J. Fehrenbach (Robert-Koch-Institut des Bundesgesundheitsamts, Berlin, Germany) for the preparation of antibodies against ACMS I and Prof. H. G. Floss (University of Washington, Seattle, WA) for generously providing ORF C, which encodes a holo-ACP synthase from S. actuosus.
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FOOTNOTES |
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* This work was supported by Grant Ke 452/8-3 from the Deutsche Forschungsgemeinschaft.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.
This paper is dedicated to Prof. Horst Kleinkauf on the occasion of his 68th birthday on November 13, 1998.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF134587 and AF134588.
To whom correspondence should be addressed. Tel.: 49-30-314-23629;
Fax: 49-30-314-73522; E-mail: Ullrich{at}chem.TU-Berlin.de.
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ABBREVIATIONS |
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The abbreviations used are: 4-MHA, 4-methyl-3-hydroxyanthranilic acid; 4-MHB, 4-methyl-3-hydroxybenzoic acid; ACMS, actinomycin synthetase; ACP, acyl carrier protein; ORF, open reading frame; PMSF, phenylmethylsulfonyl fluoride; DTE, 1,4-dithiothreitol; PAGE, polyacrylamide gel electrophoresis; 2, 3-DHB, 2,3-dihydroxybenzoic acid; PCR, polymerase chain reaction.
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REFERENCES |
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