Heterologous Expression and Structural Characterisation of a Pyrazinone Natural Product Assembly Line

Wyatt, Morgan A.; Mok, Mac C.Y.; Junop, M.S.; Magarvey, Nathan A.

doi:10.1002/cbic.201200340

Cited by 44 publications

(53 citation statements)

References 30 publications

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“…17 The lumizinones isolated from P. luminescens are structurally distinct from other 3,6-disubstituted 2(1 H )-pyrazinone metabolites, in which they are derived from a combination of tyrosine-leucine ( 1 ) or tyrosine-phenylalanine ( 2 ). The representative cyclic dipeptide phevaline was originally isolated from a terrestrial Streptomyces sp.…”

Section: Resultsmentioning

confidence: 99%

Pyrazinone protease inhibitor metabolites from Photorhabdus luminescens

Park

Crawford

2016

J Antibiot

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Photorhabdus luminescens is a bioluminescent entomopathogenic bacterium that undergoes phenotypic variation and lives in mutualistic association with nematodes of the family Heterorhabditidae. The pair infects and kills insects, and during their coordinated lifecycle, the bacteria produce an assortment of specialized metabolites to regulate its mutualistic and pathogenic roles. As part of our search for new specialized metabolites from the Photorhabdus genus, we examined organic extracts from P. luminescens grown in an amino acid rich medium based on the free amino acid levels found in the circulatory fluid of its common insect prey, the Galleria mellonella larva. Reversed-phase HPLC/UV/MS-guided fractionation of the culture extracts led to the identification of two new pyrazinone metabolites, lumizinones A (1) and B (2), together with two N-acetyl dipeptides (3 and 4). The lumizinones were produced only in the phenotypic variant associated with nematode development and insect pathogenesis. Their chemical structures were elucidated by analysis of one- and two-dimensional NMR and high-resolution ESI-QTOF-MS spectral data. The absolute configurations of the amino acids in 3 and 4 were determined by Marfey’s analysis. Compounds 1–4 were evaluated for their calpain protease inhibitory activity, and lumizinone A (1) showed inhibition with an IC50 value of 3.9 μM.

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Section: Resultsmentioning

confidence: 99%

Pyrazinone protease inhibitor metabolites from Photorhabdus luminescens

Park

Crawford

2016

J Antibiot

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“…Thus, the gut and Staphylococcus NRPSs are an example of convergent evolution toward a common scaffold, suggesting that the same compounds might play a biological role in more than one host-associated niche (Wyatt and Magarvey, 2013; Wyatt et al, 2012; Zimmermann and Fischbach, 2010). …”

Section: Discussionmentioning

confidence: 99%

Discovery of Reactive Microbiota-Derived Metabolites that Inhibit Host Proteases

Guo

Chang

Wyche

et al. 2017

Cell

193

182

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SUMMARY The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis, we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the NIH HMP stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic potential of the microbiota while avoiding the challenges of cultivating fastidious commensals.

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“…While these monodomain structures have been solved with moderate resolution (2.30 Å for NRP and 2.81 Å for AusA), the lack of bound NADPH leaves key structural and mechanistic details rather unclear (Chhabra et al, 2012;Wyatt et al, 2012). In order to define residues required for cofactor binding in MxaA R, co-crystals of MxaA R complexed with NADPH were solved by molecular replacement of the apo structure to 1.90 Å (Table S1).…”

Section: Structure Analysis Of Nadph-bound R Domainmentioning

confidence: 99%

Comprehensive Structural and Biochemical Analysis of the Terminal Myxalamid Reductase Domain for the Engineered Production of Primary Alcohols

Barajas¹,

Phelan²,

Schaub³

et al. 2015

Chemistry & Biology

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The terminal reductase (R) domain from the non-ribosomal peptide synthetase (NRPS) module MxaA in Stigmatella aurantiaca Sga15 catalyzes a non-processive four-electron reduction to produce the myxalamide family of secondary metabolites. Despite widespread use in nature, a lack of structural and mechanistic information concerning reductive release from polyketide synthase (PKS) and NRPS assembly lines principally limits our ability to redesign R domains with altered or improved activity. Here we report crystal structures for MxaA R, both in the absence and, for the first time, in the presence of the NADPH cofactor. Molecular dynamics simulations were employed to provide a deeper understanding of this domain and further identify residues critical for structural integrity, substrate binding, and catalysis. Aggregate computational and structural findings provided a basis for mechanistic investigations and, in the process, delivered a rationally altered variant with improved activity toward highly reduced substrates.

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Heterologous Expression and Structural Characterisation of a Pyrazinone Natural Product Assembly Line

Cited by 44 publications

References 30 publications

Pyrazinone protease inhibitor metabolites from Photorhabdus luminescens

Pyrazinone protease inhibitor metabolites from Photorhabdus luminescens

Discovery of Reactive Microbiota-Derived Metabolites that Inhibit Host Proteases

Comprehensive Structural and Biochemical Analysis of the Terminal Myxalamid Reductase Domain for the Engineered Production of Primary Alcohols

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