Chain Elongation, Macrolactonization, and Hydrolysis of Natural and Reduced Hexaketide Substrates by the Picromycin/Methymycin Polyketide Synthase

Given its potential role in the synthesis of novel prebiotics and applications in the pharmaceutical industry, a strong interest has developed in the enzyme levansucrase (LSC, EC 2.4.1.10). LSC catalyzes both the hydrolysis of sucrose (or sucroselike substrates) and the transfructosylation of a wide range of acceptors. LSC from the Gram-negative bacterium Erwinia tasmaniensis (EtLSC) is an interesting biocatalyst due to its high-yield production of fructooligosaccharides (FOSs). In order to learn more about the process of chain elongation, we obtained the crystal structure of EtLSC in complex with levanbiose (LBS). LBS is an FOS intermediate formed during the synthesis of longer-chain FOSs and levan. Analysis of the LBS binding pocket revealed that its structure was conserved in several related species. The binding pocket discovered in this crystal structure is an ideal target for future mutagenesis studies in order to understand its biological relevance and to engineer LSCs into tailored products.

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Characterization of the Bifunctional Glycosyltransferase/Acyltransferase Penicillin-Binding Protein 4 of Listeria monocytogenes

Journal of Bacteriology ◽

10.1128/jb.188.5.1875-1881.2006 ◽

2006 ◽

Vol 188 (5) ◽

pp. 1875-1881 ◽

Cited By ~ 27

Author(s):

Joanna Zawadzka-Skomiał ◽

Zdzislaw Markiewicz ◽

Martine Nguyen-Distèche ◽

Bart Devreese ◽

Jean-Marie Frère ◽

...

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Chain Elongation ◽

Lipid Ii ◽

Food Borne ◽

Glycan Chain ◽

Chain Polymerization ◽

Hydrolysis Of

ABSTRACT Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M−1 s−1 from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to β-lactam antibiotics but did increase the resistance of the mutant to moenomycin.

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Polyketide synthase thioesterases catalyze rapid hydrolysis of peptidyl thioesters

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2009.01.040 ◽

2009 ◽

Vol 19 (5) ◽

pp. 1413-1415 ◽

Cited By ~ 8

Author(s):

Meng Wang ◽

Peter Opare ◽

Christopher N. Boddy

Keyword(s):

Polyketide Synthase ◽

Rapid Hydrolysis ◽

Hydrolysis Of

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Brassicaceae transcriptomes reveal convergent evolution of super-accumulation of sinigrin

Communications Biology ◽

10.1038/s42003-020-01523-x ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Jinghua Yang ◽

Zhangping Li ◽

Jinmin Lian ◽

Guoning Qi ◽

Pibiao Shi ◽

...

Keyword(s):

Amino Acid ◽

Metabolic Pathways ◽

Convergent Evolution ◽

De Novo ◽

Expression Patterns ◽

Gene Clusters ◽

Chain Elongation ◽

Side Chain ◽

Crop Species ◽

Hydrolysis Of

AbstractWasabi, horseradish and mustard are popular pungent crops in which the characteristic bioactive hydrolysis of specialized glucosinolates (GSLs) occurs. Although the metabolic pathways of GSLs are well elucidated, how plants have evolved convergent mechanisms to accumulate identical GSL components remains largely unknown. In this study, we discovered that sinigrin is predominantly synthesized in wasabi, horseradish and mustard in Brassicaceae. We de novo assembled the transcriptomes of the three species, revealing the expression patterns of gene clusters associated with chain elongation, side chain modification and transport. Our analysis further revealed that several gene clusters were convergently selected during evolution, exhibiting convergent shifts in amino acid preferences in mustard, wasabi and horseradish. Collectively, our findings provide insights into how unrelated crop species evolve the capacity for sinigrin super-accumulation and thus promise a potent strategy for engineering metabolic pathways at multiple checkpoints to fortify bioactive compounds for condiment or pharmaceutical purposes.

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Evidence for an iterative module in chain elongation on the azalomycin polyketide synthase

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.12.206 ◽

2016 ◽

Vol 12 ◽

pp. 2164-2172 ◽

Cited By ~ 16

Author(s):

Hui Hong ◽

Yuhui Sun ◽

Yongjun Zhou ◽

Emily Stephens ◽

Markiyan Samborskyy ◽

...

Keyword(s):

Gene Cluster ◽

Polyketide Synthase ◽

Strong Support ◽

Artificial Chromosome ◽

Chain Extension ◽

Chain Elongation ◽

Starter Unit ◽

Polyketide Chain ◽

Streptomyces Olivaceus ◽

Streptomyces Malaysiensis

The assembly-line synthases that produce bacterial polyketide natural products follow a modular paradigm in which each round of chain extension is catalysed by a different set or module of enzymes. Examples of deviation from this paradigm, in which a module catalyses either multiple extensions or none are of interest from both a mechanistic and an evolutionary viewpoint. We present evidence that in the biosynthesis of the 36-membered macrocyclic aminopolyol lactones (marginolactones) azalomycin and kanchanamycin, isolated respectively from Streptomyces malaysiensis DSM4137 and Streptomyces olivaceus Tü4018, the first extension module catalyses both the first and second cycles of polyketide chain extension. To confirm the integrity of the azl gene cluster, it was cloned intact on a bacterial artificial chromosome and transplanted into the heterologous host strain Streptomyces lividans, which does not possess the genes for marginolactone production. When furnished with 4-guanidinobutyramide, a specific precursor of the azalomycin starter unit, the recombinant S. lividans produced azalomycin, showing that the polyketide synthase genes in the sequenced cluster are sufficient to accomplish formation of the full-length polyketide chain. This provides strong support for module iteration in the azalomycin and kanchanamycin biosynthetic pathways. In contrast, re-sequencing of the gene cluster for biosynthesis of the polyketide β-lactone ebelactone in Streptomyces aburaviensis has shown that, contrary to a recently-published proposal, the ebelactone polyketide synthase faithfully follows the colinear modular paradigm.

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Elucidating the mechanism of fluorinated extender unit loading for improved production of fluorine-containing polyketides

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614196114 ◽

2017 ◽

Vol 114 (5) ◽

pp. E660-E668 ◽

Cited By ~ 17

Author(s):

Omer Ad ◽

Benjamin W. Thuronyi ◽

Michelle C. Y. Chang

Keyword(s):

Polyketide Synthase ◽

Acyl Carrier Protein ◽

Type System ◽

Large Family ◽

Product Yield ◽

Building Blocks ◽

Chain Extension ◽

Chain Elongation ◽

Single Chain ◽

Extender Unit

Polyketides are a large family of bioactive natural products synthesized by polyketide synthase (PKS) enzyme complexes predominantly from acetate and propionate. Given the structural diversity of compounds produced using these two simple building blocks, there has been longstanding interest in engineering the incorporation of alternative extender units. We have been investigating the mechanism of fluorinated monomer insertion by three of the six different modules of the PKS involved in erythromycin biosynthesis (6-deoxyerythronolide B synthase, DEBS) to begin understanding the contribution of different steps, such as enzyme acylation, transacylation, C–C bond formation, and chain transfer, to the overall selectivity and efficiency of this process. In these studies, we observe that inactivation of acis-acyltransferase (AT) domain to circumvent its native extender unit preference leads concurrently to a change of mechanism in which chain extension with fluorine-substituted extender units switches largely to an acyl carrier protein (ACP)-independent mode. This result suggests that the covalent linkage between the growing polyketide chain and the enzyme is lost in these cases, which would limit efficient chain elongation after insertion of a fluorinated monomer. However, use of a standalonetrans-acting AT to complement modules with catalytically deficient AT domains leads to enzyme acylation with the fluoromalonyl-CoA extender unit. Formation of the canonical ACP-linked intermediate with fluoromalonyl-CoA allows insertion of fluorinated extender units at 43% of the yield of the wild-type system while also amplifying product yield in single chain-extension experiments and enabling multiple chain extensions to form multiply fluorinated products.

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Reconstitution of the FK228 Biosynthetic Pathway Reveals Cross Talk between Modular Polyketide Synthases and Fatty Acid Synthase

Applied and Environmental Microbiology ◽

10.1128/aem.01513-10 ◽

2010 ◽

Vol 77 (4) ◽

pp. 1501-1507 ◽

Cited By ~ 23

Author(s):

Shane R. Wesener ◽

Vishwakanth Y. Potharla ◽

Yi-Qiang Cheng

Keyword(s):

Fatty Acid ◽

Cross Talk ◽

Biosynthetic Pathway ◽

Fatty Acid Synthase ◽

Polyketide Synthase ◽

Fatty Acid Biosynthesis ◽

Chain Elongation ◽

E Coli ◽

Genetic Components ◽

Polyketide Chain

ABSTRACTFunctional cross talk between fatty acid biosynthesis and secondary metabolism has been discovered in several cases in microorganisms; none of them, however, involves a modular biosynthetic enzyme. Previously, we reported a hybrid modular nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) pathway for the biosynthesis of FK228 anticancer depsipeptide inChromobacterium violaceumstrain 968. This pathway contains two PKS modules on the DepBC enzymes that lack a functional acyltransferase (AT) domain, and no apparent AT-encoding gene exists within the gene cluster or its vicinity. We report here that, through reconstitution of the FK228 biosynthetic pathway inEscherichia colicells, two essential genes,fabD1andfabD2, both encoding a putative malonyl coenzyme A (CoA) acyltransferase component of the fatty acid synthase complex, are positively identified to be involved in FK228 biosynthesis. Either gene product appears sufficient to complement the AT-less PKS modules on DepBC for polyketide chain elongation. Concurrently, a gene (sfp) encoding a putative Sfp-type phosphopantetheinyltransferase was identified to be necessary for FK228 biosynthesis as well. Most interestingly, engineeredE. colistrains carrying variable genetic components produced significant levels of FK228 under both aerobic and anaerobic cultivation conditions. Discovery of thetranscomplementation of modular PKSs by housekeeping ATs reveals natural product biosynthesis diversity. Moreover, demonstration of anaerobic production of FK228 by an engineered facultative bacterial strain validates our effort toward the engineering of novel tumor-targeting bioagents.

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