Evidence for a novel phosphopantetheinyl transferase domain in the polyketide synthase for enediyne biosynthesis

The polyketide synthase associated with the biosynthesis of enediyne-containing calicheamicin contains a putative phosphopantetheinyl transferase (PPTase) domain. By cloning and expressing the C-terminal region of the polyketide synthase and in vitro phosphopantetheinylation assay, we found that the PPTase domain exhibits preferred substrate specificity towards acyl and peptidyl carrier proteins in fatty acid and non-ribosomal peptide synthesis over its cognate partner. We also found evidence suggesting that the PPTase domain adopts a pseudo-trimeric structure, distinct from the pseudo-dimeric structure of type II PPTases. The results revealed a novel type of PPTase with unique structure and substrate specificity, and suggested that the polyketide synthase probably acquired the PPTase domain from a primary metabolic pathway in evolution.     

A new substrate specificity for acyl transferase domains of the ascomycin polyketide synthase in Streptomyces hygroscopicus.

Ascomycin (FK520) is a structurally complex macrolide with immunosuppressant activity produced by Streptomyces hygroscopicus. The biosynthetic origin of C12-C15 and the two methoxy groups at C13 and C15 has been unclear. It was previously shown that acetate is not incorporated into C12-C15 of the macrolactone ring. Here, the acyl transferase (AT) of domain 8 in the ascomycin polyketide synthase was replaced with heterologous ATs by double homologous recombination. When AT8 was replaced with methylmalonyl-CoA-specific AT domains, the strains produced 13-methyl-13-desmethoxyascomycin, whereas when AT8 was replaced with a malonyl-specific domain, the strains produced 13-desmethoxyascomycin. These data show that ascomycin AT8 does not use malonyl- or methylmalonyl-CoA as a substrate in its native context. Therefore, AT8 must be specific for a substrate bearing oxygen on the alpha carbon. Feeding experiments showed that [(13)C]glycerol is incorporated into C12-C15 of ascomycin, indicating that both modules 7 and 8 of the polyketide synthase use an extender unit that can be derived from glycerol. When AT6 of the 6-deoxyerythronolide B synthase gene was replaced with ascomycin AT8 and the engineered gene was expressed in Streptomyces lividans, the strain produced 6-deoxyerythronolide B and 2-demethyl-6-deoxyerythronolide B. Therefore, although neither malonyl-CoA nor methylmalonyl-CoA is a substrate for ascomycin AT8 in its native context, both are substrates in the foreign context of the 6-deoxyerythronolide B synthase. Thus, we have demonstrated a new specificity for an AT domain in the ascomycin polyketide synthase and present evidence that specificity can be affected by context.     

Cloning, sequencing, and analysis of the griseusin polyketide synthase gene cluster from Streptomyces griseus.

A fragment of DNA was cloned from the Streptomyces griseus K-63 genome by using genes (act) for the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor as a probe. Sequencing of a 5.4-kb segment of the cloned DNA revealed a set of five gris open reading frames (ORFs), corresponding to the act PKS genes, in the following order: ORF1 for a ketosynthase, ORF2 for a chain length-determining factor, ORF3 for an acyl carrier protein, ORF5 for a ketoreductase, and ORF4 for a cyclase-dehydrase. Replacement of the gris genes with a marker gene in the S. griseus genome by using a single-stranded suicide vector propagated in Escherichia coli resulted in loss of the ability to produce griseusins A and B, showing that the five gris genes do indeed encode the type II griseusin PKS. These genes, encoding a PKS that is programmed differently from those for other aromatic PKSs so far available, will provide further valuable material for analysis of the programming mechanism by the construction and analysis of strains carrying hybrid PKS.     

Phenolic lipids synthesized by type III polyketide synthase confer penicillin resistance on Streptomyces griseus

Type III polyketide synthases (PKSs) found in plants, fungi, and bacteria synthesize a variety of aromatic polyketides. A Gram-positive, filamentous bacterium Streptomyces griseus contained an srs operon, in which srsA encoded a type III PKS, srsB encoded a methyltransferase, and srsC encoded a flavoprotein hydroxylase. Consistent with this annotation, overexpression of the srs genes in a heterologous host, Streptomyces lividans, showed that SrsA was a type III PKS responsible for synthesis of phenolic lipids, alkylresorcinols, and alkylpyrones, SrsB was a methyltransferase acting on the phenolic lipids to yield alkylresorcinol methyl ethers, and SrsC was a hydroxylase acting on the alkylresorcinol methyl ethers. In vitro SrsA reaction showed that SrsA synthesized alkylresorcinols from acyl-CoAs of various chain lengths as a starter substrate, one molecule of methylmalonyl-CoA, and two molecules of malonyl-CoA. SrsA was thus unique in that it incorporated the extender substrates in a strictly controlled order of malonyl-CoA, malonyl-CoA, and methylmalonyl-CoA to produce alkylresorcinols. An srsA mutant, which produced no phenolic lipids, was highly sensitive to beta-lactam antibiotics, such as penicillin G and cephalexin. Together with the fact that the alkylresorcinols were fractionated mainly in the cell wall fraction, this observation suggests that the phenolic lipids, perhaps associated with the cytoplasmic membrane because of their amphiphilic property, affect the characteristic and rigidity of the cytoplasmic membrane/peptidoglycan of a variety of bacteria. An srs-like operon is found widely among Gram-positive and -negative bacteria, indicating wide distribution of the phenolic lipids.     

Characterization of FdmV as an amide synthetase for fredericamycin A biosynthesis in Streptomyces griseus ATCC 43944

Fredericamycin (FDM) A is a pentadecaketide natural product that features an amide linkage. Analysis of the fdm cluster from Streptomyces griseus ATCC 43944, however, failed to reveal genes encoding the types of amide synthetases commonly seen in natural product biosynthesis. Here, we report in vivo and in vitro characterizations of FdmV, an asparagine synthetase (AS) B-like protein, as an amide synthetase that catalyzes the amide bond formation in FDM A biosynthesis. This is supported by the findings that (i) inactivation of fdmV in vivo afforded the ΔfdmV mutant strain SB4027 that abolished FDM A and FDM E production but accumulated FDM C, a biosynthetic intermediate devoid of the characteristic amide linkage; (ii) FdmV in vitro catalyzes conversion of FDM C to FDM B, a known intermediate for FDM A biosynthesis (apparent K(m) = 162 ± 67 μM and k(cat) = 0.11 ± 0.02 min(-1)); and (iii) FdmV also catalyzes the amidation of FDM M-3, a structural analog of FDM C, to afford amide FDM M-6 in vitro, albeit at significantly reduced efficiency. Preliminary enzymatic studies revealed that, in addition to the common nitrogen sources (L-Gln and free amine) of class II glutamine amidotransferases (to which AS B belongs), FdmV can also utilize L-Asn as a nitrogen donor. The amide bond formation in FDM A biosynthesis is proposed to occur after C-8 hydroxylation but before the carbaspirocycle formation.     

Properties and substrate specificity of RppA, a chalcone synthase-related polyketide synthase in Streptomyces griseus.

RppA, a chalcone synthase-related polyketide synthase (type III polyketide synthase) in the bacterium Streptomyces griseus, catalyzes the formation of 1,3,6,8-tetrahydroxynaphthalene (THN) from five molecules of malonyl-CoA. The K(m) value for malonyl-CoA and the k(cat) value for THN synthesis were determined to be 0.93 +/- 0.1 microm and 0.77 +/- 0.04 min(-1), respectively. RppA accepted aliphatic acyl-CoAs with the carbon lengths from C(4) to C(8) as starter substrates and catalyzed sequential condensation of malonyl-CoA to yield alpha-pyrones and phloroglucinols. In addition, RppA yielded a hexaketide, 4-hydroxy-6-(2',4',6'-trioxotridecyl)-2-pyrone, from octanoyl-CoA and five molecules of malonyl-CoA, suggesting that the size of the active site cavity of RppA is larger than any other chalcone synthase-related enzymes found so far in plants and bacteria. RppA was also found to synthesize a C-methylated pyrone, 3,6-dimethyl-4-hydroxy-2-pyrone, by using acetoacetyl-CoA as the starter and methylmalonyl-CoA as an extender. Thus, the broad substrate specificity of RppA yields a wide variety of products.     

Characterization and regulation of p-aminobenzoic acid synthase from Streptomyces griseus

p-Aminobenzoic acid synthase (PABA synthase) of Streptomyces griseus catalyses the conversion of chorismic acid to p-aminobenzoic acid (PABA), a precursor of the aromatic p-aminoacetophenone moiety of candicidin, a polyene macrolide antibiotic. This enzyme uses glutamine or ammonia as amino donors for PABA formation. Enzyme extracts converted [14C]chorismic acid to labelled PABA. PABA synthase was present in S. griseus IMRU 3570 only during the antibiotic producing phase. No detectable levels of the enzyme were found in cell-free extracts of nonproducing mutants of S. griseus obtained after UV mutagenesis. PABA synthase activity was found also in Streptomyces coelicolor var. aminophilus, producer of the polyene macrolide antibiotic fungimycin, but it was not present in extracts of several other streptomycetes that do not produce aromatic polyene macrolide antibiotics. PABA synthase (amidotransferase) activity was partially purified by DEAE-Bio-gel and Sephacryl S-200 filtrations. The estimated molecular weight was 50000. PABA synthase was repressed by aromatic amino acids and PABA but not by anthranilic acid. Inorganic phosphate strongly repressed but did not inhibit PABA synthase activity.     

Role of phosphopantetheinyl transferase genes in antibiotic production by Streptomyces coelicolor

The phosphopantetheinyl transferase genes SCO5883 (redU) and SCO6673 were disrupted in Streptomyces coelicolor. The redU mutants did not synthesize undecylprodigiosin, while SCO6673 mutants failed to produce calcium-dependent antibiotic. Neither gene was essential for actinorhodin production or morphological development in S. coelicolor, although their mutation could influence these processes.     

A homogeneous resonance energy transfer assay for phosphopantetheinyl transferase

Phosphopantetheinyl transferase plays an essential role in activating fatty acid, polyketide, and nonribosomal peptide biosynthetic pathways, catalyzing covalent attachment of a 4′-phosphopantetheinyl group to a conserved residue within carrier protein domains. This enzyme has been validated as an essential gene to primary metabolism and presents a target for the identification of antibiotics with a new mode of action. Here we report the development of a homogeneous resonance energy transfer assay using fluorescent coenzyme A derivatives and a surrogate peptide substrate that can serve to identify inhibitors of this enzyme class. This assay lays a blueprint for translation of these techniques to other transferase enzymes that accept fluorescent substrate analogues.     

Analysis of Streptomyces coelicolor phosphopantetheinyl transferase, AcpS, reveals the basis for relaxed substrate specificity

The transfer of the phosphopantetheine chain from coenzyme A (CoA) to the acyl carrier protein (ACP), a key protein in both fatty acid and polyketide synthesis, is catalyzed by ACP synthase (AcpS). Streptomyces coelicolor AcpS is a doubly promiscuous enzyme capable of activation of ACPs from both fatty acid and polyketide synthesis and catalyzes the transfer of modified CoA substrates. Five crystal structures have been determined, including those of ligand-free AcpS, complexes with CoA and acetyl-CoA, and two of the active site mutants, His110Ala and Asp111Ala. All five structures are trimeric and provide further insight into the mechanism of catalysis, revealing the first detailed structure of a group I active site with the essential magnesium in place. Modeling of ACP binding supported by mutational analysis suggests an explanation for the promiscuity in terms of both ACP partner and modified CoA substrates.     

Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis.

The genes encoding the polyketide synthase (PKS) portion of the niddamycin biosynthetic pathway were isolated from a library of Streptomyces caelestis NRRL-2821 chromosomal DNA. Analysis of 40 kb of DNA revealed the presence of five large open reading frames (ORFs) encoding the seven modular sets of enzymatic activities required for the synthesis of a 16-membered lactone ring. The enzymatic motifs identified within each module were consistent with those predicted from the structure of niddamycin. Disruption of the second ORF of the PKS coding region eliminated niddamycin production, demonstrating that the cloned genes are involved in the biosynthesis of this compound.     

Myo-inositol-1-phosphate synthase in different Streptomyces griseus variants

The activity of myo-inositol-1-phosphate synthase (MIPS, EC 5.5.1.4.) from streptomycin producing and non-producing strains of Streptomyces griseus was measured during the life cycle on different culture media. The activity varied in the different S. griseus variants and depended on the time of cultivation and the composition of the culture medium. Strains characterized by low MIPS levels are also low streptomycin producers. The enzyme is unstable. It loses 70% of its activity in the crude extract after 18 h at 0 degree C. (NH4)2SO4 and DMSO (dimethyl sulfoxide) in 20% solution are suitable stabilizing agents, the loss of activity being only 10% in 0.5 M (NH4)2SO4 solution during 24 h. With the applied purification procedure the specific activity of the enzyme was increased 23-fold. According to preliminary estimations, its molecular mass (Mr) is about 216 kDa with a pH optimum of 8.3 and Mg2+-dependent enzyme activity.     

A phosphopantetheinyl transferase that is essential for mitochondrial fatty acid biosynthesis

In this study we report the molecular genetic characterization of the Arabidopsis mitochondrial phosphopantetheinyl transferase (mtPPT), which catalyzes the phosphopantetheinylation and thus activation of mitochondrial acyl carrier protein (mtACP) of mitochondrial fatty acid synthase (mtFAS). This catalytic capability of the purified mtPPT protein (encoded by AT3G11470) was directly demonstrated in an in vitro assay that phosphopantetheinylated mature Arabidopsis apo‐mtACP isoforms. The mitochondrial localization of the AT3G11470‐encoded proteins was validated by the ability of their N‐terminal 80‐residue leader sequence to guide a chimeric GFP protein to this organelle. A T‐DNA‐tagged null mutant mtppt‐1 allele shows an embryo‐lethal phenotype, illustrating a crucial role of mtPPT for embryogenesis. Arabidopsis RNAi transgenic lines with reduced mtPPT expression display typical phenotypes associated with a deficiency in the mtFAS system, namely miniaturized plant morphology, slow growth, reduced lipoylation of mitochondrial proteins, and the hyperaccumulation of photorespiratory intermediates, glycine and glycolate. These morphological and metabolic alterations are reversed when these plants are grown in a non‐photorespiratory condition (i.e. 1% CO₂ atmosphere), demonstrating that they are a consequence of a deficiency in photorespiration due to the reduced lipoylation of the photorespiratory glycine decarboxylase.     

Glutathione S-Transferase Isoenzymes from Streptomyces griseus

An inducible, cytosolic glutathione S-transferase (GST) was purified from Streptomyces griseus. GST isoenzymes with pI values of 6.8 and 7.9 used standard GST substrates including 1-chloro-2,4-dinitrobenzene. GST had subunit and native M_rs of 24 and 48, respectively, and the N-terminal sequence SMILXYWDIIRGLPAH.     

Histidine ammonia-lyase from Streptomyces griseus.

Histidine ammonia-lyase (histidase; HutH) has been purified to homogeneity from Streptomyces griseus and the N-terminal amino acid (aa) sequence used to clone the histidase-encoding structural gene, hutH. The purified enzyme shows typical saturation kinetics and is inhibited competitively by D-histidine and histidinol phosphate. High concentrations of K.cyanide inactivate HutH unless the enzyme is protected by the substrate or histidinol phosphate. On the basis of the nucleotide sequence, the hutH structural gene would encode a protein of 53 kDa with an N terminus identical to that determined for the purified enzyme. Immediately upstream from hutH is a region that strongly resembles a class of Streptomyces promoters active during vegetative growth; however, there is no obvious ribosome-binding site adjacent to the hutH translation start codon. The deduced aa sequence of an upstream partial open reading frame shows no similarity with other proteins, including HutP of Bacillus subtilis and HutU of Pseudomonas putida. Promoter-probe analysis indicates that promoter activity maps within the DNA surrounding the hutH start codon. Pairwise comparisons of the primary structures of bacterial and mammalian histidases, together with the unique kinetic properties and gene organization, suggest that streptomycete histidase may represent a distinct family of histidases.     

An endogalactosaminidase from Streptomyces griseus.

An endogalactosaminidase has been purified 34-fold from the culture filtrate of Streptomyces griseus. This enzyme cleaves GalN-GalN linkages in oligogalactosaminoglycan, a galactosamine-rich oligosaccharide isolated from the culture filtrate of a Neurospora mutant. Since some or all of the GalN-GalN bonds in this molecule link positions 1 and 4, and are in the alpha-configuration, we are probably dealing with an endo-alpha-(1 leads to 4)-galactosaminidase, bu this characterization is only tentative because the few bonds cleaved by the enzyme could have a different structure. The enzyme is inactive towards N-acetyl-oligogalactosaminoglycan and chitosan. The endogalactosaminidase preparations also cleave high molecular weight galactosaminoglycan (obtained from Neurospora) into fragments greater than or equal to 10(4) daltons in molecular weight, and catalyze the release of Neurospora sporelings from the glass surfaces to which they are anchored. Galactosaminoglycan-cleaving and sporeling-releasing activities elute jointly from DEAE-cellulose columns. This observation provides further support for an earlier proposal that the sporelings are anchored to the glass by means of galactosaminoglycan molecules.     

Natural biocombinatorics in the polyketide synthase genes of the actinobacterium Streptomyces avermitilis

Modular polyketide synthases (PKSs) of bacteria provide an enormous reservoir of natural chemical diversity. Studying natural biocombinatorics may aid in the development of concepts for experimental design of genes for the biosynthesis of new bioactive compounds. Here we address the question of how the modularity of biosynthetic enzymes and the prevalence of multiple gene clusters in Streptomyces drive the evolution of metabolic diversity. The phylogeny of ketosynthase (KS) domains of Streptomyces PKSs revealed that the majority of modules involved in the biosynthesis of a single compound evolved by duplication of a single ancestor module. Using Streptomyces avermitilis as a model organism, we have reconstructed the evolutionary relationships of different domain types. This analysis suggests that 65% of the modules were altered by recombinational replacements that occurred within and between biosynthetic gene clusters. The natural reprogramming of the biosynthetic pathways was unambiguously confined to domains that account for the structural diversity of the polyketide products and never observed for the KS domains. We provide examples for natural acyltransferase (AT), ketoreductase (KR), and dehydratase (DH)–KR domain replacements. Potential sites of homologous recombination could be identified in interdomain regions and within domains. Our results indicate that homologous recombination facilitated by the modularity of PKS architecture is the most important mechanism underlying polyketide diversity in bacteria.