Specificity of Nonribosomal Peptide Synthetases in the Biosynthesis of the <i>Pseudomonas</i> <i>virulence factor</i>

Morgan, Gina L.; Kretsch, Ashley M; Maria, Kevin C. Santa; Weeks, Savannah J; Ли, Бо

doi:10.1021/acs.biochem.9b00360

Cited by 22 publications

(48 citation statements)

References 34 publications

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“…In view of the structures of 2 and 1 , it was hypothesized that l ‐valine ( 3 ) was the substrate for their biosyntheses. Our suggestion is in agreement with a recent study performed on PvfC, a homologue of HamD initially discovered in a Pseudomonas entomophila strain . Li and co‐workers identified the key residues of the active site, and demonstrated through bioinformatics and experimental studies that 3 is the initial substrate of the PvfC NRPS.…”

Section: Methodssupporting

confidence: 92%

“…However, because of the structure of fragin, we proposed that either valine or leucine would be the substrate of the HamD adenylation domain. This is supported by recently published data that identified valine as the amino acid substrate of PvfC, which has in its adenylation domain a ten‐amino‐acid code identical to that in HamD . To test valine incorporation into fragin, feeding experiments were performed with [U‐ 13 C 5 ]‐ l ‐valine ( 4 , Scheme ) and the supernatant was analyzed by HRMS.…”

Section: Methodsmentioning

confidence: 57%

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Biosynthesis and Structure–Activity Relationship Investigations of the Diazeniumdiolate Antifungal Agent Fragin

et al. 2020

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Only a few natural products incorporating a diazeniumdiolate moiety have been isolated, and these compounds usually display a broad range of biological activities. Only recently has the first diazeniumdiolate natural product biosynthetic gene cluster been identified in Burkholderia cenocepacia H111, which produces the fungicide (−)‐fragin and the signal molecule rac‐valdiazen. In this study, l‐valine was identified as the initial substrate of (−)‐fragin biosynthesis with the aid of feeding experiments using isotopically labelled amino acid. The formation of the diazeniumdiolate was chemically studied with several proposed intermediates. Our results indicate that the functional group is formed during an early stage of the biosynthesis. Furthermore, an oxime compound was identified as a degradation product of (−)‐fragin and was also observed in the crude extract of the wild‐type strain. Moreover, a structure–activity relationship analysis revealed that each moiety of (−)‐fragin is essential for its biological activity.

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Section: Methodssupporting

confidence: 92%

Section: Methodsmentioning

confidence: 57%

Biosynthesis and Structure–Activity Relationship Investigations of the Diazeniumdiolate Antifungal Agent Fragin

et al. 2020

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“…This result indicated that it may be the indigenous signal produced by Pss ( Figure 2C) and is also in line with a bioinformatics analysis of the substrate specificity of the adenylation domain of MgoA. 37 To increase the sensitivity of our detection strategy, we developed a UHPLC-MS/MS method using our synthetic standard. An extraction was performed on two liters culture media containing Pss and its ΔmgoA mutant.…”

Section: Inactivation Of Leudiazensupporting

confidence: 78%

“…27,34 Furthermore, in 2018, Li and coworkers engineered a P. entomophila strain to overexpress the pvf cluster leading to the production of three N-oxide metabolites 35 and they investigated the role of the N-oxygenase in the biosynthesis of valdiazen (1). 36 In a follow up study, the same research group investigated the substrate specificity of the NRPS domain of PvfC and its homologs MgoA and HamD in different proteobacteria and concluded that at least two types of signaling molecules are produced by the different strains 37…”

Section: Introductionmentioning

confidence: 99%

A Volatile Signal Controls Virulence in the Plant PathogenPseudomonas syringaepv.syringaeand a Strategy for Infection Control in Organic Farming

Sieber

Mathew

Jenul

et al. 2020

Preprint

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Pseudomonas syringae is an important pathogen of many agriculturally valuable crops. Among the various pathovars described P. syringae pv. syringae (Pss) has a particularly wide host range, infecting primarily woody and herbaceous host plants. The ability of Pss to cause bacterial apical necrosis of mango trees is dependent on the production of the antimetabolite toxin mangotoxin. The production of this toxin was shown to be regulated by a self-produced signaling molecule. In this study, we determined the structure of the Pss signal molecule belonging to the recently described family of diazeniumdiolate communication molecules. Employing a targeted mass spectrometry-based approach, we provide experimental evidence that the major signal produced by Pss is the volatile compound leudiazen, which controls mangotoxin production and virulence in a detached tomato leaflet infection model. Experimental results demonstrate that KMnO4 solution inactivates leudiazen and that treatment of infected leaves with KMnO4 abolishes necrosis. This strategy represents the first example of chemically degrading a signaling molecule to interfere with bacterial communication. The application of KMnO4 solution, which is regulatorily approved in organic farming, may constitute an environmentally friendly strategy to control Pss infections.

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“…We sought to determine the role of PvfB in (d)PNO biosynthesis. Valine is incorporated into (d)PNOs by PvfC from P. entomophila L48 (Figure 1 B), [1,4] but PvfB does not modify free valine or 2,5-diisopropyl pyrazine (Supporting Information, Figure S8), thus, we hypothesized that PvfB directly N-oxygenates PvfC-tethered valine. We used liquid chromatography-coupled high-resolution mass spectrometry (LC-HRMS) to study the activity of PvfB on PvfC-tethered valine.…”

mentioning

confidence: 99%

In Vitro Reconstitution Reveals a Central Role for the N‐Oxygenase PvfB in (Dihydro)pyrazine‐N‐oxide and Valdiazen Biosynthesis

Morgan

Ли

2020

Angew Chem Int Ed

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The Pseudomonas virulence factor (pvf) operon is essential for the biosynthesis of two very different natural product scaffolds: the (dihydro)pyrazine-N-oxides and the diazeniumdiolate, valdiazen. PvfB is a member of the nonheme diiron N-oxygenase enzyme family that commonly convert anilines to their nitroaromatic counterparts. In contrast, we show that PvfB catalyzes N-oxygenation of the aamine of valine, first to the hydroxylamine and then the nitroso, while linked to the carrier protein of PvfC. PvfB modification of PvfC-tethered valine was observed directly by protein NMR spectroscopy, establishing the intermediacy of the hydroxylamine. This work reveals a central role for PvfB in the biosynthesis of (dihydro)pyrazine-N-oxides and valdiazen. ThePseudomonas virulence factor (pvf, Figure 1 A), a widely conserved operon in proteobacteria, [1] plays important roles in virulence and bacterial cell-to-cell signaling. [2] This cluster, first reported from Pseudomonas entomophila L48, has now been identified in more than 500 strains (Supporting Information, Dataset S1). [1] We showed that pvf-encoded enzymes from P. entomophila L48 produce a novel family of molecules-(dihydro)pyrazine-N-oxides [(d)PNOs, Figure 1 B)]. Concurrently, the ham operon, a pvf-like cluster from Burkholderia cenocepacia H111, was implicated in the production of a very different molecule, valdiazen-a valinol diazeniumdiolate (Figure 1 B). [3] The (d)PNOs and valdiazen both contain unusual NO functionalities but differ in overall structure. We set out to understand the biosynthetic chemistries for these different compounds. Pvf commonly comprises four genes: a nonribosomal peptide synthetase (NRPS, pvfC or hamD), a non-heme diiron enzyme (pvfB or hamC), and two genes of unknown function, pvfA/hamA and pvfD/hamE (Figure 1 A). The ham operon contains a fifth gene, hamB, encoding a putative cupin protein. Both pvfC and pvfB from P. entomophila are necessary to produce the (d)PNOs. [4] NRPSs selectively activate and tether amino or carboxylic acids to thiolation domains where substrates undergo modifications by tailoring enzymes. [5] The NPRS PvfC activates and tethers l-valine to its thiolation domain (Figure 1 B), [1, 4] and similar chemistry could be predicted for HamD. The role of the putative diiron N-oxygenases PvfB or HamC, however, remained unclear.

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Specificity of Nonribosomal Peptide Synthetases in the Biosynthesis of the Pseudomonas virulence factor

Cited by 22 publications

References 34 publications

Biosynthesis and Structure–Activity Relationship Investigations of the Diazeniumdiolate Antifungal Agent Fragin

Biosynthesis and Structure–Activity Relationship Investigations of the Diazeniumdiolate Antifungal Agent Fragin

A Volatile Signal Controls Virulence in the Plant PathogenPseudomonas syringaepv.syringaeand a Strategy for Infection Control in Organic Farming

In Vitro Reconstitution Reveals a Central Role for the N‐Oxygenase PvfB in (Dihydro)pyrazine‐N‐oxide and Valdiazen Biosynthesis

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