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Originally published In Press as doi:10.1074/jbc.M604895200 on August 8, 2006

J. Biol. Chem., Vol. 281, Issue 40, 29660-29668, October 6, 2006
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A Dedicated Phosphopantetheinyl Transferase for the Fredericamycin Polyketide Synthase from Streptomyces griseus*

Yong Huang{ddagger}, Evelyn Wendt-Pienkowski{ddagger}, and Ben Shen{ddagger}§1

From the {ddagger}Division of Pharmaceutical Sciences, the §University of Wisconsin National Cooperative Drug Discovery Group, and the Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53705

Received for publication, May 22, 2006 , and in revised form, August 2, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polyketide synthases cannot be functional unless their apo-acyl carrier proteins (apo-ACPs) are post-translationally modified by covalent attachment of the 4'-phosphopantetheine group to the highly conserved serine residue, and this reaction is catalyzed by phosphopantetheinyl transferases (PPTases). Cloning and sequence analysis of the 33-kb fredericamycin (FDM) biosynthetic gene cluster from Streptomyces griseus revealed fdmW, whose deduced gene product showed significant sequence homology to known PPTases. Biochemical characterization of FdmW in vitro confirmed that it is a PPTase. Inactivation of fdmW resulted in ~93% reduction of FDM production, and complementation of the fdmW::aac (3)IV mutant by expressing fdmW in trans restored FDM production to a level comparable with that of the wild-type strain. Although FdmW can phosphopantetheinylate various ACPs, it prefers its cognate substrate, the FdmH ACP, with a Km of 5.8 µM and a kcat/Km of 8.1 µM–1·min–1, to heterologous ACPs, such as the TcmM ACP with a Km of 1.0 x 102 µM and a kcat /Km of 0.6 µM–1·min–1. These findings suggest that FdmW is specific for FDM biosynthesis. FdmW therefore represents the first holo-ACP synthase-type PPTase identified from an aromatic polyketide biosynthetic gene cluster.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The covalently attached 4'-phosphopantetheine prosthetic group in various carrier proteins, such as acyl carrier proteins (ACPs)2 in fatty acid synthases (FASs) and polyketide synthases (PKSs) and peptidyl carrier proteins in nonribosomal peptide synthetases (NRPSs), activates the acyl or peptidyl substrate by a thioester linkage and provides a flexible "swinging arm" to channel the growing intermediates in these multienzyme complexes or multifunctional enzymes (1, 2). The transfer of 4'-phosphopantetheine from coenzyme A (CoA) to the conserved serine residue of the carrier proteins is catalyzed by a phosphopantetheinyl transferase (PPTase), which recognizes the conserved GX(D/E/H)S motif (Fig. 1A). This post-translational modification converts the apo-carrier proteins to the functional holo-carrier proteins. Phosphopantetheinylated carrier proteins are also involved in many other biological processes, such as the incorporation of D-alanine into D-alanyl-lipoteichoic acid in Gram-positive organisms (3), the biosynthesis of lipid A (4), and lysine biosynthesis in yeast (5).

Various PPTases have been found from bacteria, fungi, and humans, and they can be classified into three groups based on their sequence similarity and substrate specificity toward carrier proteins (6). The holo-ACP synthase (ACPS)-type PPTases are present in the genome sequences of almost every bacterium and are typically 120 amino acids in length. ACPSs from Escherichia coli (7), Streptococcus pneumoniae (8), Bacillus subtilis (6), and Streptomyces coelicolor (9), are capable of phosphopantetheinylating apo-ACPs from FASs. ACPSs are believed to be involved in fatty acid biosynthesis and are essential to the host organisms, as exemplified by dpj gene inactivation in E. coli by using mini-Tn10 transposons (10). The ACPS-type PPTases can also modify apo-ACPs from PKSs in vitro but not the peptidyl carrier proteins from NRPS pathways.

The second type of PPTases, the Sfp-type PPTases, is often approximately twice the size of the ACPS-type and can phosphopantetheinylate both ACPs and peptidyl carrier proteins. In bacteria, this type of PPTase genes has been found both clustered with PKS or NRPS gene clusters, such as sfp from the surfactin biosynthesis pathway in B. subtilis (11) and mta for myxothiazol biosynthesis in myxobacteria (12), and independent of the PKS or NRPS gene clusters, such as svp from Streptomyces verticillus, the producer of antitumor antibiotic bleomycin (13). PPTase genes associated with PKS or NRPS gene clusters, such as sfp or mta, are often dispensable for polyketide or peptide biosynthesis, whereas the true physiological role for stand-alone PPTase genes, such as svp, remains to be established. Marahiel and co-workers (6, 14) have recently suggested that the Sfp-type PPTases can also serve as PPTases for fatty acid biosynthesis in B. subtilis and Pseudomonas aeruginosa in the absence of ACPS-type PPTases. Current evidence is also consistent with the belief that a single Sfp-type PPTase with broad substrate specificity exists in humans (15).


Figure 1
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  		FIGURE 1.
A, PPTase-catalyzed post-translational modification, transferring the 4'-phosphopantetheine moiety from CoA to a conserved Ser residue of the apo-carrier protein to produce the holo-carrier protein. B, proposed pathway for FDM biosynthesis featuring the FDM type II PKS. The step catalyzed by FdmW is boxed.

 
The third group of PPTases is found at the C-terminal domain of yeast FAS. They activate the apo-ACP domain in FAS in cis by self-phosphopantetheinylation (16). Little is known about this type of PPTases because they have only recently been discovered.

Bacterial aromatic PKSs, also known as type II PKSs, minimally consist of four enzymes for polyketide biosynthesis, including a holo-ACP, two ketoacyl-ACP synthase subunits KS{alpha} and KSbeta (also known as chain length factor), and a malonyl CoA:ACP transacylase. They synthesize a broad range of aromatic polyketides, many of which have important pharmacological properties, such as the anticancer drugs doxorubicin and mithramycin and the antibiotic tetracycline. The post-translational modification of the apo-ACP to form the holo-ACP is essential for the proper function of type II PKS and must be catalyzed by a PPTase. Mutation of the conserved serine residue in the actinorhodin ACP (S42A) completely inactivated the PKS, confirming the conserved Ser as the site of phosphopantetheinylation (17).

Some aromatic polyketide gene clusters do not contain PPTase genes, such as the ones for the biosynthesis of actinorhodin, tetracenomycin, doxorubicin, mithramycin, and tetracycline. To explain this curiosity, it has been suggested that the ACPS associated with the host FAS could modify both the FAS and PKS apo-ACPs (18). This hypothesis is supported by the relatively broad substrate specificity of ACPS toward ACPs, the successful interchange of the actinorhodin ACP gene with the ACP gene from the Saccharopolyspora erythraea FAS (17), as well as the structure similarity of FAS ACPs and actinorhodin ACP established by an NMR study (19).

In contrast, putative Sfp-type PPTase genes are located in other type II PKS gene clusters, such as those for the biosynthesis of granaticin, griseorhodin, jadomycin, landomycin, medermycin, resistomycin, and simocyclinone (20–27), suggesting that they are dedicated to a particular biosynthetic pathway. Furthermore, inactivation of the jadM PPTase gene caused a 95–97% decrease in jadomycin yield, which implies that no other PPTase in the organism was able to modify the jadomycin ACP efficiently in vivo (23). Substrate promiscuity or specificity of PPTases therefore has to be considered in any attempt to engineer polyketide or peptide biosynthetic machinery for structural diversity in heterologous hosts because effective PPTase-catalyzed modification of carrier proteins is crucial for functional overexpression of the PKS or NRPS gene clusters (28, 29).

During an investigation of the biosynthesis of the antitumor antibiotic fredericamycin (FDM) A in Streptomyces griseus, we cloned and sequenced the fdm biosynthetic genes encoding a type II PKS and confirmed its function by gene inactivation and complementation in S. griseus and heterologous expression in Streptomyces albus (Fig. 1B) (30). Among the genes identified within the fdm cluster, the deduced product of fdmW was found to have significant sequence similarity to known ACPS-type PPTases. We chose to address the role of fdmW in FDM biosynthesis and here report the results of in vivo and in vitro characterization of the FdmW PPTase. Our findings establish that FdmW is a PPTase dedicated to FDM biosynthesis, and FdmW therefore represents the first example of an ACPS-type PPTase identified from an aromatic polyketide biosynthetic gene cluster.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
General Procedures—E. coli DH5{alpha}, E. coli BL21 (DE3) (Novagen, Madison, WI), E. coli TB1 (New England Biolabs, Beverly, MA), E. coli M15(pREP4) (Qiagen), and S. griseus ATCC49344 (30) were used in this work. pBS18 (13), pBS25 (13), pBS3024 (31), pBS4012 (30), pCK12 (32), pSET151 (33), pWHM3 (34), and pWHM1250 (34) were described previously. pET28a (Novagen), pSP72 (Promega, Madison, WI), pMAL-c2x (New England Biolabs), and pSPORT1 (Invitrogen) were from commercial sources. Plasmid preparation was carried out using commercial kits (Qiagen). Streptomyces chromosomal DNA was isolated according to literature protocols (35). Restriction enzymes and other molecular biology reagents were from commercial sources, and DNA digestion and ligation followed standard methods (36). For Southern analysis, digoxigenin labeling of DNA probes, hybridization, and detection were performed according to the protocols provided by the manufacturer (Roche Applied Science). Automated DNA sequencing was provided by University of Wisconsin-Madison Biotechnology Center (Madison, WI), and the data were analyzed using PE Biosystems version 3.7 of Sequencing Analysis. Bioinformatics analysis, such as the sequence alignment and comparison, and the isoelectric point (pI) calculation was performed using the GCG package provided by BioComp research computing service in University of Wisconsin-Madison.

Inactivation and Complementation of fdmW in S. griseus—The fdm biosynthetic gene cluster has been mapped to two overlapping cosmids, pBS4029 and pBS4030. To inactivate fdmW, a 5.6-kb PstI fragment containing fdmW from pBS4030 was first cloned into the same site of pSPORT1 to afford pBS4031. A 1.5-kb BamHI DNA fragment of the aac (3)IV apramycin resistance gene (35) was then inserted into the NotI site of fdmW in pBS4031 to afford pBS4032. The mutated fdmW::acc (3)IV gene was finally moved from pBS4032 as a 7.1-kb PstI fragment into PstI of pSET151 to yield pBS4033. pBS4033 was introduced into the S. griseus wild-type strain by transformation, and the double cross-over mutant SB4008 was obtained through an established protocol (30) and then confirmed by Southern analysis.

To construct the fdmW complementation construct, a 750-bp KpnI-XhoI fragment of the fdmW gene from pBS4031 was cloned into pSPORT1 at the KpnI-SalI sites to gain cloning sites. The fdmW gene was then excised as a PstI-HindIII fragment and ligated into the same sites of either pWHM1250 or pWHM3 to yield pBS4034 or pBS4035, respectively. The expression of fdmW in the former construct would be under the control of both ErmE* (35) and its native promoters, whereas that in the latter construct would be under the control of its native promoter only. pBS4034 and pBS4035 as well as pWHM3 as a negative control were introduced separately into the SB4008 mutant strain by transformation to generate, respectively, strains SB4009 (SB4008/pBS4034), SB4010 (SB4008/pBS4035), and SB4011 (SB4008/pWHM3). Production, isolation, HPLC, and mass spectrometry analysis of the fermentation culture of S. griseus wild-type and recombinant (i.e. SB4008, SB4009, SB4010, and SB4011) strains were carried out as described previously (30).

Overproduction and Purification of DEBS1-ACP1, FdmH, FdmW, LnmJ-ACP5, Svp, and TcmM—The DEBS1-ACP1 gene was amplified by PCR from plasmid pCK12, which harbors the full-length DEBS1 gene, using 5'-CCGGAATTCCATATGCTGTTCGAGC-3' as a forward primer (the EcoRI site is underlined) and a reverse primer 5'-CGCGCAAGCTTGAGTTCGGCGGCCCAGGTG-3' (the HindIII site is underlined). The resultant PCR product was sequenced to verify PCR fidelity, digested with EcoRI-HindIII, and cloned into the same sites of pSP72 to produce pBS4036. The DEBS1-ACP1 gene was finally excised as a 0.3-kb NdeI-HindIII fragment from pBS4036 and cloned into the same sites of pET28a to yield pBS4037. The latter resulted in the overproduction of DEBS1-ACP1 as a fusion protein with His6 tags at both the C and N termini.

The fdmH gene was amplified by PCR from pBS4012 using a forward primer 5'-CCGGAATTCCATATGAGCACCATCAGCTTCAACGAC-3' (the EcoRI site is underlined) and a reverse primer 5'-GCGCGCAAGCTTGACCCCCGCCAGGCTGGT-3' (the HindIII site is underlined). The resultant PCR product was sequenced to verify PCR fidelity, digested with EcoRI-HindIII, and cloned into the same sites of pSP72 to produce pBS4038. The fdmH gene was finally moved from pBS4038 as a 0.3-kb NdeI-HindIII fragment into the same sites of pET28a to afford pBS4039. The latter resulted in the overproduction of FdmH as a fusion protein with His6 tags at both the C and N termini.

The fdmW gene was amplified by PCR from pBS4040, a pSPORT1-based plasmid with a 1,749-bp KpnI-XhoI DNA fragment containing fdmW gene subcloned from pBS4030, using a forward primer 5'-CCGGAATTCCATATGCAACCTGACGACGAGGGC-3' (the EcoRI site is underlined) and a reverse primer 5'-GCGCGCAAGCTTGGCCGCACCGACGACGAC-3' (the HindIII site is underlined). The resultant PCR product was sequenced to verify PCR fidelity, digested with EcoRI-HindIII, and cloned into the same sites of pSP72 to yield pBS4041. The fdmW gene was finally cloned from pBS4041 as a 0.5-kb NdeI-HindIII fragment into the same sites of pET28a to yield pBS4042. The latter resulted in the overproduction of FdmW as a fusion protein with His6 tags at both the C and N termini.

Alternatively, the fdmW gene was amplified from pBS4040 with the same forward primer but a different reverse primer with the fdmW stop codon 5'-GATAAGCTTTCAGGCCGCACCGACGACGAC-3' (the HindIII site is underlined). The resultant PCR product was sequenced to verify PCR fidelity, digested with EcoRI-HindIII, and cloned into the same sites of pMal-c2x to generate pBS4043. The latter resulted in the overproduction of FdmW as an N-terminal maltose-binding protein (MBP) fusion protein.

To generate the N-terminal truncated FdmW, the following forward primer 5'-GATGAATTCCATATGATCGTGGGTGTGGGGATC-3' (the EcoRI site is underlined) and reverse primer 5'-GATAAGCTTTCAGGCCGCACCGACGAC-3' (the HindIII site is underlined) were used for the PCR amplification. The resultant PCR product was sequenced to verify PCR fidelity, digested with EcoRI-HindIII, cloned into the same sites of pMAL-c2x to afford pBS4044. The latter resulted in the production of a truncated FdmW (i.e. deletion of 37 amino acids, QPDDEGAREW RRGPGGSGRF PWPLRLRARH PRPAPPR, at its N terminus) as an N-terminal MBP fusion protein.

For overproduction of the DEBS1-ACP1 and FdmH proteins, E. coli BL21 (DE3) was transformed with pBS4037 and pBS4039, respectively. The resultant transformants were grown in LB at 37 °C for 5 h. A 2–5 ml portion of this culture was used to inoculate 500 ml of LB, and incubation continued at 37 °C until the A595 nm reached 0.6. Isopropyl-beta-D-thiogalactopyranoside (0.1 mM) was then added, and incubation continued at 30 °C for 5 h. The cells were harvested, first treated with lysozyme, and then disrupted by sonication. DEBS1-ACP1 or FdmH was purified by affinity chromatography on Ni2+-NTA in 50 mM sodium phosphate buffer, pH 8.0, 300 mM NaCl, and eluted with 250 mM imidazole in the same buffer. The eluted protein fractions were collected, dialyzed against 1 mM EDTA, 1 mM DTT, 10% glycerol, 10 mM Tris·HCl, pH 8.0, and stored at –80 °C for in vitro assays. The isolated yield for DEBS1-ACP1 or FdmH was estimated to be 2 or 18 mg/liter, respectively.

To obtain pure apo-FdmH for kinetic assays, the FdmH purified via Ni2+-NTA affinity chromatography was further subjected to an anion exchange chromatography on a MonoQ column (10 x 10 cm; Amersham Biosciences) to remove the small amount of holo-FdmH co-purified by Ni2+-NTA affinity chromatography. The column was eluted with a 20-min linear gradient from 0 to 1.0 M NaCl in 50 mM Tris·HCl (pH 8.0) at a flow rate of 1 ml/min. The fractions containing apo-FdmH were pooled, dialyzed against 1 mM EDTA, 1 mM DTT, 10% glycerol, 10 mM Tris·HCl, pH 8.0, and stored at –80 °C for in vitro assays.

For overproduction of FdmW as a His6-tagged fusion protein, pBS4042 was similarly introduced into E. coli BL21 (DE3) as described above. Although FdmW was well produced, it was completely insoluble under all conditions tested. For production of FdmW and its truncated form as MBP fusion proteins, E. coli TB1 was transformed with pBS4043 or pBS4044, respectively. The resultant transformants were grown, induced, harvested, and disrupted, and the crude cell free extract was prepared as described above, except for the omission of imidazole in the buffer. Purification of the MBP fusion proteins on amylose resin was carried out in 50 mM sodium phosphate buffer, pH 8.0, following the protocol recommended by the manufacturer (New England Biolabs). The fractions containing FdmW or the truncated FdmW were pooled, dialyzed against 1 mM EDTA, 1 mM DTT, 10% glycerol, 10 mM Tris·HCl, pH 8.0, and stored at –80 °C for in vitro assays. The isolated yields for FdmW and its truncated form as MBP fusion proteins were estimated to be 3 and 2 mg/liter, respectively. LnmJ-ACP5 (31), Svp (13), and TcmM (13) were overproduced and purified as described previously.

The protein concentrations were determined based on the calculated extinction coefficients at 280 nm: DEBS1-ACP1, 1,320 M–1 cm–1; FdmH, 3,840 M–1 cm–1; FdmW, 87,480 M–1 cm–1; truncated FdmW, 77,350 M–1 cm–1; LnmJ-ACP5, 5,960 M–1 cm–1; Svp, 34,615 M–1 cm–1; and TcmM, 2,980 M–1 cm–1.

Phosphopantetheinylation of ACPs by Svp and FdmW in Vitro and Kinetic Analysis—The pH profile of FdmW was determined in 75 mM MES/sodium acetate (for pH 4.5–6.5) and 75 mM Tris·HCl (for pH 7.0–9.5) buffers. FdmW exhibited an optimal pH at approximately 8.0; thus all in vitro studies of FdmW were carried out at pH 8.0. For in vitro phosphopantetheinylation of ACPs, reaction mixtures contained 10–50 µM apo-ACPs (i.e. DEBS1-ACP1, FdmH, LnmJ-ACP5, or TcmM), 0.25 µM Svp or 0.2 µM FdmW, 250 µM CoA, 5 mM DTT, 12.5 mM MgCl2, 75 mM Tris·HCl, pH 8.0, in a final volume of 50 µl. The assays were initiated by the addition of CoA, incubated at 37 °C for 15–60 min, and terminated by the addition of EDTA to a final concentration of 50 mM and then frozen immediately at –80 °C. To follow phosphopantetheinylation of the apo-ACPs by HPLC analysis, the samples were injected to an analytic Jupiter C-18 column (5 µm, 300 Å, 250 x 4.6 mm; Phenomenex, Belmont, CA), eluted with a 20-min linear gradient from 15% CH3CN to 90% CH3CN in H2O with 0.1% CF3CO2H at a flow rate of 1 ml/min. Peaks corresponding to apo- and holo-FdmH were collected, lyophilized, and dissolved in H2O for electrospray ionization-mass spectrometry (MS) analysis, which was performed on an Aglient (Palo Alto, CA) 1000 HPLC-MSD SL instrument.

For kinetic analysis, the reaction mixtures contained 2–320 µM apo-ACPs, 25 nM Svp or 20 nM FdmW, 250 µM CoA, 5 mM DTT, 12.5 mM MgCl2, 75 mM Tris·HCl, pH 8.0, in a final volume of 100 µl. The assays were initiated, incubated at 37 °C for 5 min, and terminated and analyzed by HPLC as described above. The amount of converted holo-ACPs was calculated based on the peak abundance of the apo-ACPs and holo-ACPs under initial velocity conditions. Kinetic parameters were calculated by the standard nonlinear regression method.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
FdmW Is an Unusual ACPS-type PPTase by Sequence Analysis—The product of the fdmW gene was assigned to be a putative PPTase based on the significant sequence similarity with the ACPS-type PPTases from various Streptomyces species and other bacteria (Table 1). FdmW contains the two conserved motifs VGID and FAAKEAVAK found in the PPTase superfamily: (V/I)G(V/I)D and (F/W)XXKE(A/S)hhK (Fig. 2), in which Asp and Glu are involved in chelating the metal cofactor Mg2+, and Phe is essential for binding the phosphopantetheinyl moiety of holo-ACP as part of a hydrophobic pocket, as shown by the co-crystal structure of B. subtilis ACPS and ACP (37). FdmW also has some unusual characteristics compared with other ACPS-type PPTases. Although most of these enzymes are ~120 amino acids in length, FdmW has a long N-terminal region upstream of its VGID motif, which is seen elsewhere only in putative ACPSs from B. melitensis (38) and Streptomyces resistomycificus (26) (Fig. 2). This N-terminal region is ~37 amino acids in length and rich in Arg (nine) and Pro (seven) residues (Fig. 2). Consequently, FdmW is a very basic protein; its predicated isoelectric point (pI) is 12.04, greatly above the pIs of other ACPS-type PPTases, which typically range from 5.81 to 10.46 (Table 1). The RemD ACPS (pI of 10.46) from S. resistomycificus has one of the closest pIs to FdmW, perhaps suggesting that these two PPTases function in a very similar manner.


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  		TABLE 1
Sequence comparison of the FdmW PPTase with selected ACPS-type and Sfp-type PPTases

Sequence similarity and identity and pI calculation are carried out using the GCG package provided by BioComp research computing service at University of Wisconsin-Madison.

 


Figure 2
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  		FIGURE 2.
Sequence comparison of FdmW from S. griseus with selected ACPS-type PPTases from B. subtilis (CAB12269), E. coli (P24224), S. coelicolor (NP_628902), S. pneumoniae (AAG22706), and S. resistomycificus (CAE51176). Given in parentheses are the protein accession numbers. The two conserved motifs for PPTases are shaded, and the Arg- and Pro-rich (both shaded) N terminus of FdmW is boxed.

 
Inactivation of the fdmW Gene and Complementation of the fdmW::aac (3)IV Mutant—To establish a physiological role for FdmW in S. griseus, fdmW was inactivated by a standard gene replacement method using pSET151 as the carrier vector (36). The gene replacement plasmid pBS4033 was constructed by insertion of the apramycin resistance gene aac (3)IV into fdmW followed by cloning the mutated fdmW::aac (3)IV fragment into pSET151. After introduction of pBS4033 into the S. griseus wild-type strain by selection for apramycin resistance, replacement of the wild-type fdmW was achieved by double cross-over homologous recombination (Fig. 3A). The genotype of the resultant mutant, named SB4008, was confirmed by Southern analysis of total DNAs from the S. griseus wild-type and mutant strains with a 0.6-kb KpnI-NotI DNA probe that includes the fdmW locus. Digestion of the genomic DNA by MluI resulted in detection of the predicted band shift from 2.9 to 1.9 kb in wild-type versus mutant strains (Fig. 3B).

The ability of the wild-type fdmW gene to complement the inactivated fdmW::aac (3)IV gene in vivo was tested in the S. griseus SB4008 mutant. Two versions of fdmW complementation plasmids were constructed: pBS4034 was a pWHM1250 derivative with the constitutively expressed strong promoter ErmE* placed before the fdmW gene and its 205-bp upstream region presumably containing its native promoter, whereas pBS4035 was a pWHM3 derivative that did not contain the ErmE* promoter. These two plasmids as well as the pWHM3 vector as a negative control (note that pWHM1250 was originally derived from pWHM3 (34)) were introduced into individual isolates of the S. griseus SB4008 mutant strain, and transformants resistant to apramycin and thiostrepton, designated as SB4009 (i.e. SB4008/pBS4034), SB4010 (i.e. SB4008/pBS4035), and SB4011 (i.e. SB4008/pWHM3), were selected and cultured. Extracts from the fermentation culture of S. griseus wild-type and SB4008, SB4009, SB4010, and SB4011 recombinant strains were analyzed for FDM production by HPLC (Fig. 3C). Although no adverse effect on the growth characteristics of S. griseus SB4008 was observed, inactivation of fdmW resulted in ~93% reduction of FDM production in SB4008. FDM production was restored to the wild-type level in strains SB4009 and SB4010, and the identity of FDM in the latter strains was confirmed by electrospray ionization-MS analysis, yielding a m/z at 538 for [M-H]– (calculated [M-H]– for FDM, C30H21O9, is 538). These data confirmed that the large reduction of FDM production in the SB4008 strain was due to the inactivation of fdmW, which plays an important role in FDM biosynthesis.


Figure 3
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  		FIGURE 3.
Characterization of fdmW by gene inactivation and fdmW::aac (3)IV mutant complementation. A, schematic representation for the isolation of the fdmW::aac (3)IV mutant strain SB4008 via double cross-over homologous recombination. B, BamHI; K, KpnI; M, MluI; N, NotI; P, PstI. B, Southern analysis of S. griseus wild-type (lane 1) and SB4008 (lane 2) mutant strain genomic DNAs digested with MluI using the 0.6-kb KpnI-NotI DNA fragment of fdmW as a probe. C, HPLC analysis of FDM A ({diamondsuit}) production isolated from recombinant strains SB4009 (panel I), SB4010 (panel II), SB4011 (panel III), and SB4008 (panel IV) with the wild-type strain (panel V) as a control.

 
Production and Purification of FdmW and Its Putative Substrates from E. coli—To characterize FdmW in vitro, we expressed fdmW and purified the overproduced FdmW protein from E. coli. The fdmW gene was highly expressed in E. coli using the pET expression system under control of the T7 lac promoter (pBS4042) but only as inclusion bodies under all conditions tested, including low temperature (16–22 °C) and low or no isopropyl-beta-D-thiogalactopyranoside induction. Expression of fdmW in pMAL-c2x as an N-terminal MBP fusion protein (pBS4043) avoided this problem. The MBP-FdmW fusion protein was purified using amylose affinity chromatography. It migrated in SDS-PAGE with a molecular mass of ~60 kDa, consistent with the calculated molecular mass of 59,807 (Fig. 4, lane 4). Because there is an "extra" N-terminal region in FdmW compared with other ACPS-type PPTases, an N-terminally truncated version of the MBP-FdmW was also produced (pBS4044) and purified (Fig. 4, lane 3).

The FdmW substrate specificity was tested with four ACPs from two types of bacterial PKSs. FdmH, the putative ACP of FDM biosynthesis, and TcmM of tetracenomycin biosynthesis are ACPs from type II PKSs. The LnmJ-ACP5 from the hybrid PKS/NRPS of leinamycin biosynthesis and the DEBS1-ACP1 from the 6-deoxyerthronolide B synthase (DEBS) of erythromycin biosynthesis are ACPs of type I PKSs. TcmM (13) and LnmJ-ACP5 (31) were produced and purified as previously described. The fdmH gene was cloned into the pET-28a expression vector (pBS4039), and FdmH was well produced as a fusion protein with the His6 tags at both the N and C termini and readily purified by Ni2+-NTA affinity chromatography. The gene encoding the first ACP in module 1 of DEBS (DEBS1) was also cloned into pET28a and expressed (pBS4037), and the overproduced DEBS1-ACP1 as a fusion protein with His6 tags at both the N and C termini was similarly purified by Ni2+-NTA affinity chromatography. Upon SDS-PAGE, DEBS1-ACP1 and FdmH migrated with the expected molecular mass of ~10–12 kDa (calculated molecular mass, 10,963 for DEBS1-ACP1 and 12,725 for FdmH) (Fig. 4, lanes 1 and 2).

Phosphopantetheinylation of ACPs by FdmW and Svp PPTases—An HPLC assay was used to determine FdmW as a PPTase in vitro and its substrate specificity toward different ACPs. Svp, a promiscuous PPTase that is known to be able to phosphopantetheinylate the TcmM ACP and LnmJ-ACP5 was used as a control (13, 31). When such PKS ACPs are produced in E. coli, they are partially phosphopantetheinylated by the endogenous PPTases, most likely the constitutively produced ACPS for fatty acid biosynthesis. TcmM and LnmJ-ACP5 purified by affinity chromatography on Ni2+-NTA resin showed two peaks, which corresponded to their apo- and holo-forms (Fig. 5, A and B, panels I). Incubation of TcmM with CoA in the presence of Svp led to the disappearance of the apo-TcmM, consistent with the conversion of apo- to its holo-TcmM form (Fig. 5A, panel II). The increase in the amount of holo-TcmM after incubation with CoA and FdmW establishes that FdmW is a PPTase that can post-translationally modify the TcmM ACP (Fig. 5A, panel III). When either Svp or FdmW was incubated with the LnmJ-ACP5 or DEBS1-ACP1, Svp converted essentially all apo-LnmJ-ACP5 and ~70% apo-DEBS-ACP1 to their holo-forms, whereas FdmW converted ~10% apo-LnmJ-ACP5 and 12% DEBS1-ACP1 to their holo-forms (Fig. 5, B and C). These results together indicate that the FdmW PPTase, albeit less efficiently than Svp, shows some promiscuity toward different ACPs from both type I and type II PKSs.

As noted above, overproduction of FdmH from E. coli similarly resulted in both the holo- and apo-forms (Fig. 5D, panel I). Incubation of FdmH with Svp or FdmW in the presence of CoA resulted in conversion of apo-FdmH into its holo-form (Fig. 5D, panels II and III). The molecular identity of both the apo- and holo-forms of FdmH was confirmed by electrospray ionization-MS analysis: holo-FdmH yielded a mass of 13,063 Da (calculated, 13,065 Da), a 340 mass unit increase over the apo-FdmH that was measured with a mass of 12,722 Da (calculated 12725 Da). The N-terminal truncated FdmW was also tested for its phosphopantetheinylation activity toward apo-FdmH and apo-TcmM. In both cases, no holo-ACP conversion could be detected, suggesting that the 37-amino acid N terminus of FdmW is necessary for the PPTase activity under the conditions used.


Figure 4
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  		FIGURE 4.
Purification of the FdmW PPTase and ACPs. Lane 1, DEBS1-ACP1; lane 2, FdmH; lane 3, MBP-FdmW (N terminus truncated); lane 4, MBP-FdmW; lane 5, molecular mass markers.

 


Figure 5
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  		FIGURE 5.
HPLC analysis of in vitro 4'-phosphopantetheinylation of apo-ACPs from selected type I and type II PKSs by FdmW in comparison with Svp. A, TcmM; B, LnmJ-ACP5; C, DEBS1-ACP1, and D, FdmH. Panels I, apo-ACPs alone; panels II, apo-ACPs with Svp as positive controls; and panels III, apo-ACPs with FdmW. {triangledown}, apo-ACPs; •, holo-ACPs. Carrier protein and PPTase concentrations used were: 50 µM TcmM, 40 µM LnmJ-ACP5, 10 µM DEBS-ACP1, 50 µM FdmH, and 0.25 µM Svp or 0.2 µM FdmW, respectively. The assays for TcmM and FdmH were incubated for 15 min, whereas the assays for LnmJ-ACP5 and DEBS1-ACP1 were incubated for 60 min.

 
Kinetic Characterization of FdmW and Svp toward FdmH and TcmM—The catalytic efficiency of FdmW and Svp toward FdmH and TcmM was determined by the HPLC based method for comparison. Although TcmM overproduced in E. coli was purified essentially in the apo-form, the ratio of apo-FdmH to holo-FdmH purified from E. coli was ~85:15, necessitating further purification by anion chromatography to afford pure apo-FdmH (Fig. 6A). The pH profile of FdmW as a PPTase was determined prior to kinetic analysis; it had an optimal pH of 8.0 with FdmH as its substrate, much higher than the optimal pHs observed for other PPTases, such as Sfp (pH 6.0), Svp (pH 6.5–7.0), and S. pneumoniae ACPS (pH 6.5) (8, 13, 39). Initial velocity assays were used to determine the kinetic constants for FdmW and Svp catalyzed phosphopantetheinyl transfer from CoA to FdmH or TcmM, and the Michaelis-Menten equation was used to fit the experimental data sets (Figs. 6, B and C). The kinetic constants of FdmW and Svp are summarized in Table 2. FdmW shows a k of 47 or 57 min–1 61 and 35 min–1 and Km values of 167 and 4.7 µM toward FdmH and TcmM, respectively, suggesting that FdmH is a much poorer substrate toward Svp in comparison with TcmM.


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  		TABLE 2
Kinetic constants of FdmW and Svp PPTases towards apo-FdmH and apo-TcmM ACPs

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We identified the fdmW gene from the fdm biosynthetic gene cluster and, overproduced, purified, and characterized its gene product in this study (Fig. 1). Sequence analysis showed that fdmW encodes an unusual ACPS-type PPTase, with an extended N-terminal region rich in Arg and Pro (Fig. 2) as well as a high pI (Table 1). Gene inactivation and complementation of fdmW support the conclusion that fdmW is not involved in fatty acid biosynthesis, which is essential for the viability of streptomycetes under most growth conditions (35), but it plays an important role in FDM biosynthesis (Fig. 3). FdmW, produced as a soluble protein in the form of an N-terminal MBP fusion, was capable of converting various apo-ACPs to holo-ACPs from either modular type I PKSs or iterative type II PKSs (Figs. 4 and 5). This characteristic is typical of several other ACPS-type PPTases that show broad substrate specificity toward ACPs. Removal of the unique N terminus of FdmW resulted in the loss of its PPTase activity. Kinetic analysis established that FdmW has higher catalytic efficiency toward its cognate substrate FdmH as compared with TcmM from the tetracenomycin PKS (Table 2). Although it exhibits very similar kcat for FdmH and TcmM, FdmW has a Km value 17 times lower for FdmH than TcmM. Comparison of the second order rate constant using FdmH as a substrate shows that FdmW is 22-fold more efficient than Svp toward its cognate substrate. These results together suggest that FdmH is unique, requiring a dedicated PPTase, FdmW, for its post-translational modification, a conclusion that also agrees well with the finding that FdmH is a poor substrate for Svp compared with TcmM (the catalytic efficiency for FdmH dropped ~19-fold in comparison with TcmM).


Figure 6
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  		FIGURE 6.
Kinetic characterization of FdmW as a PPTase in comparison with Svp. A, HPLC analysis of purified apo-TcmM (panel I) and apo-FdmH (panel II) and initial velocities (V) of in vitro 4'-phosphopantetheinylation of apo-ACPs catalyzed by FdmW (B) and Svp (C) in concentration [E] as function of apo-ACPs. •, apo-FdmH; {blacksquare}, apo-TcmM.

 
The characterization of FdmW represents the first ACPS-type PPTase identified from an aromatic polyketide biosynthetic gene cluster. FDM PKS catalyzes the biosynthesis of a pentadecaketide, a polyketide with the longest chain length characterized to date (30) (Fig. 1B). It is therefore tempting to speculate that the FdmH ACP may have uniquely evolved to accommodate the longer polyketide intermediate, and likewise a dedicated PPTase was also evolved. The findings that FdmW exhibits high specificity toward FdmH and that FdmH serves as a poor substrate to known promiscuous PPTases such as Svp are consistent with the above hypothesis.

One possible speculation for the inability of other endogenous but non-cognate PPTases to substitute for FdmW in FDM biosynthesis in vivo may be the presence of specific interactions between the unusual N-terminal region of FdmW and FdmH. The N-terminal Arg residues of FdmW could form additional salt bridges with acidic residues of FdmH, which are vital to the functioning of the FdmW-FdmH complex. The crystal structure of the B. subtilis ACPS and ACP complex revealed that, although their contacts were mostly hydrophilic, three Arg residues from the ACPS play crucial roles by forming salt bridges with the acidic residues Glu or Asp in the ACP, which facilitates the interaction between the helix {alpha}1 of ACPS and the helix {alpha}3 of ACP (37). Another explanation could be that the electrostatic interaction between FdmW and FdmH is much stronger than that of the FAS ACPS from S. griseus and FdmH because of the highly basic nature of FdmW and the acidic nature of FdmH, which has a pI of 3.53, lower than the pIs of the FAS ACPs from S. coelicolor (pI 3.89), S. glauscences (pI 3.82), and Streptomyces avermitilis (pI 3.79). This reasoning is consistent with the observation by Walsh and co-workers (41) that the more negatively charged ACPs are better substrates for E. coli ACPS (pI 9.98) because of the existence of the stronger electrostatic interaction.

In conclusion, FdmW represents the first example of an ACPS-type PPTase for an aromatic type II PKS system. The discovery and characterization of such a PPTase as FdmW is critical to the continued expansion of the toolbox available for genetic engineering and combinatorial biosynthesis for the discovery of new natural products.


   FOOTNOTES
 
* This work is supported in part by National Institutes of Health Grants CA35381 and CA113297. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of National Institutes of Health Independent Scientist Award AI51687. To whom correspondence should be addressed: Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison, 777 Highland Ave., Madison, WI 53705. Tel.: 608-263-2673; Fax: 608-262-5345; E-mail: bshen{at}pharmacy.wisc.edu.

2 The abbreviations used are: ACP, acyl carrier protein; ACPS, holo-acyl carrier protein synthase; CoA, coenzyme A; MS, mass spectrometry; FAS, fatty acid synthase; FDM, fredericamycin; MBP, maltose-binding protein; NRPS, nonribosomal peptide synthetase; PKS, polyketide synthase; PPTase, phosphopantetheinyl transferase; HPLC, high pressure liquid chromatography; NTA, nitrilotriacetic acid; DTT, dithiothreitol; MES, 2-(N-morpholino)ethanesulfonic acid; DEBS, 6-deoxyerthronolide B synthase. Back


   ACKNOWLEDGMENTS
 
We thank the Analytical Instrumentation Center of the School of Pharmacy, UW-Madison for support in obtaining MS data, Dr. Hao Jiang for assistance in constructing pBS4033 and isolating the SB4008 mutant, and Dr. C. Richard Hutchinson for critical reading of the manuscript.



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 DISCUSSION
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