Functional analysis of putative beta-ketoacyl:acyl carrier protein synthase and acyltransferase active site motifs in a type II polyketide synthase of Streptomyces glaucescens

Meurer, Guido; Hutchinson, C. Richard

doi:10.1128/jb.177.2.477-481.1995

Cited by 36 publications

(37 citation statements)

References 38 publications

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“…The domain has the sequence GHS, which is conserved throughout all actinomycete PKS KS homologs (11) and which is also a motif found at the active site of acyltransferases. Recent experimental evidence, obtained by mutagenesis of the actinorhodine and the tetracenomycin KS, has cast doubt on whether the serine at the center of the putative acyltransferase domain is essential for PKS function (24,32).…”

Section: Discussionmentioning

confidence: 99%

Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant

1995

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Streptomyces coelicolor A3(2) synthesizes each half molecule of the dimeric polyketide antibiotic actinorhodin (Act) from one acetyl and seven malonyl building units, catalyzed by the Act polyketide synthase (PKS). The synthesis is analogous to fatty acid biosynthesis, and there is evident structural similarity between PKSs of Streptomyces spp. and fatty acid synthases (FASs). Each system should depend on a malonyl coenzyme A:acyl carrier protein malonyltransferase, which charges the FAS or PKS with the malonyl units for carbon chain extension. We have purified the Act acyl carrier protein-dependent malonyltransferase from stationary-phase, Act-producing cultures and have determined the N-terminal amino acid sequence and cloned the structural gene. The deduced amino acid sequence resembles those of known malonyltransferases of FASs and PKSs. The gene lies some 2.8 Mb from the rest of the act cluster, adjacent to an open reading frame whose gene product resembles ketoacylsynthase III of Escherichia coli FAS. The malonyltransferase was expressed equally as well during vegetative growth (when other components of the act PKS were not expressed) as in the stationary phase, suggesting that the malonyltransferase may be shared between the FAS and PKS of S. coelicolor. Disruption of the operon containing the malonyltransferase gene proved to be impossible, supporting the idea that the malonyltransferase plays an essential role in fatty acid biosynthesis.The aromatic polyketide actinorhodin (Act) (Fig. 1) is a secondary metabolite produced by Streptomyces coelicolor A3(2), genetically the most studied member of the genus. A C 16 carbon chain forms the backbone of each half-molecule of Act and is synthesized from one acetyl coenzyme A (acetyl CoA) starter and seven malonyl CoA extender units. These units are condensed together (with loss of CO 2 ) in a sequential fashion by the Act polyketide synthase (PKS) (14), a process which shows clear similarities to the mechanism of fatty acid biosynthesis (21, 33). The linear intermediate then undergoes cyclizations and further, ''tailoring'' modifications to yield the final molecule. A set of genes (act) involved in the synthesis of actinorhodin has been identified and located on one continuous (22-kb) section of the S. coelicolor chromosome (28). The act cluster has been completely sequenced (4,(10)(11)(12)16); it contains a set of some 22 genes that include the structural genes for the Act PKS, genes which encode the enzymes involved in the subsequent modification of the PKS product, and genes for activation of expression of the act cluster and for actinorhodin export.The act PKS itself consists of a series of discrete open reading frames (ORFs) encoding several proteins that share considerable amino acid sequence similarity with the components of Escherichia coli fatty acid synthase (FAS) (11). These are (i) a ketosynthase (KS), which catalyzes the condensation of the acyl building units; (ii) an acyl carrier protein (ACP), which serves a dual purpose as the recipient for the ...

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Section: Discussionmentioning

confidence: 99%

Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant

1995

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“…S. glaucescens WMH1077 T , S. lividans 1326, and the plasmids pWHM3 (70), pWHM860 (47), pWHM752 (60), and pWHM769 (60) are described elsewhere. The ermE p * promoter was obtained as described earlier (47,60). The TA PCR cloning vector was purchased from Invitrogen (San Diego, Calif.).…”

Section: Methodsmentioning

confidence: 99%

A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms

et al. 1997

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To examine as randomly as possible the role of the ␤-ketoacyl and acyl carrier protein (ACP) components of bacterial type II polyketide synthases (PKSs), homologs of the chain-length-factor (CLF) genes were cloned from the environmental community of microorganisms. With PCR primers derived from conserved regions of known ketosynthase (KS ␣ ) and ACP genes specifying the formation of 16-to 24-carbon polyketides, two CLF (KS ␤ ) genes were cloned from unclassified streptomycetes isolated from the soil, and two were cloned from soil DNA without the prior isolation of the parent microorganism. The sequence and deduced product of each gene were distinct from those of known KS ␤ genes and, by phylogenetic analysis, belonged to antibiotic-producing PKS gene clusters. Hybrid PKS gene cassettes were constructed with each novel KS ␤ gene substituted for the actI-ORF2 or tcmL KS ␤ subunit genes, along with the respective actI-ORF1 or tcmK KS ␣ , tcmM ACP, and tcmN cyclase genes, and were found to produce an octaketide or decaketide product characteristic of the ones known to be made by the heterologous KS ␣ gene partner. Since substantially less than 1% of the microorganisms present in soil are thought to be cultivatable by standard methods, this work demonstrates a potential way to gain access to a more extensive range of microbial molecular diversity and to biosynthetic pathways whose products can be tested for biological applications.The polyketide metabolites are a large group of structurally complex and diverse natural products, many of which are clinically valuable antibiotics or chemotherapeutic agents or have other useful pharmacological activities (48). Polyketides are synthesized by polyketide synthases (PKSs) which, like the related fatty acid synthases, are multifunctional enzyme assemblies that catalyze repeated decarboxylative condensations between enzyme-bound acylthioesters (52). The structural diversity of polyketides is a result of the different numbers and types of acyl units involved, coupled with various possibilities for enzyme-mediated reduction, dehydration, cyclization, and aromatization of the initial ␤-ketoacyl condensation products (52). Manipulation of the sequence or specificity of the PKS enzymes can lead to the production of novel secondary metabolites (28,29,33). The key to further exploitation of this approach to molecular diversity lies in an increased understanding of the genetics and biochemistry of the various PKS systems.Recent progress in the cloning and sequencing of microbial PKS genes has led to the identification of at least two architecturally different types of PKSs (28, 33). Modular type I PKSs, represented by erythromycin A (11, 17), avermectin (41), rapamycin (58), and soraphen (57), are discrete multifunctional enzymes sporting numerous unique active site domains. The domains are grouped into distinct modules that in turn control the sequential addition of acylthioester units to the growing polyketide chain and the subsequent modification of the respective condensation prod...

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“…To prepare pWHM411, a 0.69-kb NruI-BamHI fragment that contains 40 bp of upstream sequence and the entire coding region of the dnrN gene was cloned into pWHM860 (28) between the ermE* promoter and a terminator.…”

Section: Methodsmentioning

confidence: 99%

The DnrN protein of Streptomyces peucetius, a pseudo-response regulator, is a DNA-binding protein involved in the regulation of daunorubicin biosynthesis

Furuya

Hutchinson

1996

J Bacteriol

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Actinomycetes are gram-positive bacteria that possess an interesting life cycle involving morphological differentiation (aerial mycelia and spore formation) and specialized secondary metabolism (antibiotic production). To regulate these complex processes, the bacteria appear to have evolved an intricate regulatory hierarchy with several different mechanisms. Some of these processes are unique to the Streptomyces spp., such as the bldA gene (20), and others are well-known in eukaryotes, such as hormone-like molecules (31) and serine-threonine or tyrosine kinases (24). Other mechanisms resemble ones common in prokaryotes. An increasingly important example of the latter is the two-component system (4, 17, 46, 50), which consists of a histidine kinase that undergoes autophosphorylation in response to environmental or developmental changes and then transfers its phosphate group to an aspartyl residue of a response regulator protein (34,35,40).In our study of the biosynthesis of daunorubicin (DNR) and doxorubicin (DXR), commercially important antitumor drugs (Fig. 1), sequence analysis of the dnrN gene (see Fig. 2) showed that its product is very similar to response regulator proteins of the FixJ subfamily (13,32). The N-terminal region of DnrN has a putative phosphorylation site (D-55) and other conserved sites required for activation by phosphorylation and characteristic of response regulators. However, the D-553E (D55E) and D-553N (D55N) substitutions at the putative site of phosphorylation resulted in a reduction rather than a complete loss of DnrN activity (32). Furthermore, analysis of the 60-kb region sequenced around dnrN has not uncovered a putative sensor kinase gene, which often resides near the response regulator gene. Since DnrN lacks two key characteristics of canonical response regulators, we call it a pseudo-response regulator.Our previous results from mutant complementation (32) and transcriptional analysis (23) experiments showed that dnrN is essential for transcription of the dnrI regulatory gene and functions epistatically with respect to dnrI. DnrI, whose sequence is similar to other Streptomyces regulatory proteins such as AfsR (16), RedD (30), and actII-Orf4 (9), in turn activates the DNR and DXR structural and resistance genes (23). Among streptomycetes, this is a clear case in which a putative response regulator gene is known to govern expression of antibiotic biosynthesis and resistance genes in such a direct and essential manner.In spite of considerable information about response regulator proteins in other bacteria (1,11,37,48), so far none of them have been characterized from streptomycetes. Here we report that the DnrN protein binds specifically to the dnrI promoter region and is not likely to require phosphorylation for its binding. We also show that DNR has a positive effect on the DnrN level in Streptomyces peucetius and suggest how DNR could feedback repress dnrI expression.

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Functional analysis of putative beta-ketoacyl:acyl carrier protein synthase and acyltransferase active site motifs in a type II polyketide synthase of Streptomyces glaucescens

Cited by 36 publications

References 38 publications

Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant

Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant

A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms

The DnrN protein of Streptomyces peucetius, a pseudo-response regulator, is a DNA-binding protein involved in the regulation of daunorubicin biosynthesis

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