Repositioning of a Domain in a Modular Polyketide Synthase to Promote Specific Chain Cleavage

Cortés, Jesús M.; Wiesmann, Kirsten E.H.; Roberts, Gareth A.; Brown, Murray J. B.; Staunton, James; Leadlay, Peter F.

doi:10.1126/science.7770773

Cited by 257 publications

(177 citation statements)

References 22 publications

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“…In particular the carboxyl-terminal portion of deoxyerythronolide B synthase, encoding the thioesterase-cyclase function that is responsible for the detachment of deoxyerythronolide B synthase, was repositioned in an internal region of the complex to generate a shorter triketide lactone (25).…”

Section: Discussionmentioning

confidence: 99%

Engineering of Peptide Synthetases

Ferra¹,

Rodriguez²,

Tortora³

et al. 1997

Journal of Biological Chemistry

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Peptide synthetases are large enzymatic complexes that catalyze the synthesis of biologically active peptides in microorganisms and fungi and typically have an unusual structure and sequence. Peptide synthetases have recently been engineered to modify the substrate specificity to produce peptides of a new sequence. In this study we show that surfactin synthetase can also be modified by moving the carboxyl-terminal intrinsic thioesterase region to the end of the internal amino acid binding domains, thus generating strains that produce new truncated peptides of the predicted sequence. Omission of the thioesterase domain results in nonproducing strains, thus showing the essential role of this region and the possibility of obtaining peptides of different lengths by genetic engineering. Secretion of the peptides depends on the presence of a functional sfp gene.Two mechanisms for the biosynthesis of peptides are known to exist in bacteria and fungi, ribosomal synthesis and production by peptide synthetases. These are large enzymatic complexes responsible for the synthesis of hundreds of types of peptides, some of which have immunoactive, antibiotic, antifungal, or surfactant properties. Whereas polypeptides produced by ribosomal synthesis typically contain only the amino acids directly specified by the triplets of the genetic code, peptides built on synthetases often contain unusual amino or hydroxy acids as building blocks that are not present in proteins. The amino acids can be modified by peptide synthetases either through methylation, hydroxylation, or enantiomerization. The peptides are typically short (up to about 20 residues) and can be linear, circular, or branched (1, 2). Peptide synthesis proceeds by the "multiple carrier thiotemplate mechanism" (3, 4), and many of the details of these systems remain to be investigated experimentally. According to this mechanism each domain recognizes a specific amino (or hydroxy) acid that is covalently bound to the cofactor via a thioester bond after activation to the corresponding acyladenylate derivative. The growth of the polypeptide chain thus occurs through a series of thioester bond cleavages and the simultaneous formation of amide or ester bonds in the peptide. At the end of synthesis of each peptide, the chain is thought to be released from the enzyme by a thioesterase (TE) 1 activity encoded in the synthetase gene (1). Peptide synthetases are interesting not only from a scientific and evolutionary point of view but also for their biotechnological potential. Many enzymatically synthesized peptides are in fact biologically active, and some of them are industrially produced, among which are cyclosporins, surfactin, and fungicides. A growing number of laboratories are involved in applied research projects focused on the isolation and characterization of new peptide synthetases and on the genetic manipulation of peptide synthetase genes to optimize peptide production and to genetically modify the sequence of the peptides produced. In fact, the structural organization...

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Section: Discussionmentioning

confidence: 99%

Engineering of Peptide Synthetases

Ferra¹,

Rodriguez²,

Tortora³

et al. 1997

Journal of Biological Chemistry

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“…Hopefully, the approach described here, which was developed to enable a broader investigation of the type II PKSs, will complement other, newly emerging approaches to antibiotic discovery (28). These include several genome projects involving known pathogens (20) to provide additional targets for conventional screening programs and de novo drug design, new strategies in combinatorial biology (12,31,32,45,65,66) to yield novel scaffolding for decorative (bio-)chemistry, and attempts to access the immense marine biodiversity (30).…”

Section: Discussionmentioning

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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“…The potential diversity of macrocyclic compounds attainable by TE catalysis is further amplified by the fact that a myriad of macrocyclization reactions are performed by TE domains from different NRPS and polyketide synthetase assembly lines (2). The utility of such terminal TE domains can be exploited in efforts to engineer synthetases to generate novel compounds (28)(29)(30)(31). Additionally, it is feasible that these small (25-35 kDa) enzymatic domains in isolation could be used in the generation of diverse libraries of macrocyclic compounds with possible biological activity in areas distinct from the natural synthetase product.…”

Section: Discussionmentioning

confidence: 99%

The thioesterase domain from a nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides

Kohli

Takagi

Walsh

2002

Proc. Natl. Acad. Sci. U.S.A.

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Nonribosomal peptide synthetases responsible for the production of macrocyclic compounds often use their C-terminal thioesterase (TE) domain for enzymatic cyclization of a linear precursor. The excised TE domain from the nonribosomal peptide synthetase responsible for the production of the cyclic decapeptide tyrocidine A, TycC TE, retains autonomous ability to catalyze head-to-tail macrocyclization of a linear peptide thioester with the native sequence of tyrocidine A and can additionally cyclize peptide analogs that incorporate limited alterations in the peptide sequence. Here we show that TycC TE can catalyze macrocyclization of peptide substrates that are dramatically different from the native tyrocidine linear precursor. Several peptide thioesters that retain a limited number of elements of the native peptide sequence are shown to be substrates for TycC TE. These peptides were designed to integrate an Arg-Gly-Asp sequence that confers potential activity in the inhibition of ligand binding by integrin receptors. Although enzymatic hydrolysis of the peptide thioester substrates is preferred over cyclization, TycC TE can be used on a preparative scale to generate both linear and cyclic peptide products for functional characterization. The products are shown to be inhibitors of ligand binding by integrin receptors, with cyclization and N ␣ -methylation being important contributors to the nanomolar potency of the best inhibitors of fibrinogen binding to ␣IIb␤3 integrin. This study provides evidence for TycC TE as a versatile macrocyclization catalyst and raises the prospect of using TE catalysis for the generation of diverse macrocyclic peptide libraries that can be probed for novel biological function.M acrocyclic structure is a desirable feature for biologically or pharmaceutically active compounds. Macrocyclization stabilizes products from degradation and decreases the conformational flexibility of a molecule compared with its linear analog by constraining it to a biologically active conformation (1). In the biosynthesis of macrocyclic natural products, such as the macrolactam antibiotic tyrocidine, the macrolactone antifungal fengycin, and the macrolactone siderophore enterobactin by nonribosomal peptide synthetases (NRPSs), a C-terminal thioesterase (TE) domain introduces this constraint by catalyzing cyclization of a linear precursor (2, 3). Analogous cyclases in polyketide synthetase assemblies generate erythromycin and ketolide antibiotics as well as other bioactive macrolactones (4, 5).We have recently shown that several 28-to 35-kDa C-terminal TE domains excised from megadalton NRPS subunit proteins retain autonomous macrocyclization activity with peptide thioester substrates that mimic their natural protein-bound peptide phosphopantetheinyl substrates (6, 7). As a representative example, TycC TE, the C-terminal TE domain from the synthetase responsible for the biosynthesis of tyrocidine, macrocyclizes a soluble decapeptide thioester with the native peptide sequence of the antibiotic tyrocidine A (Fig. ...

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Repositioning of a Domain in a Modular Polyketide Synthase to Promote Specific Chain Cleavage

Cited by 257 publications

References 22 publications

Engineering of Peptide Synthetases

Engineering of Peptide Synthetases

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 thioesterase domain from a nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides

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