Direct Acylation of Carrier Proteins with Functionalized β-Lactones

Amoroso, Jon W.; Borketey, Lawrence S.; Prasad, Gitanjeli; Schnarr, Nathan A.

doi:10.1021/ol100684s

Cited by 15 publications

(19 citation statements)

References 20 publications

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“…An alternate approach was employed which relied upon a direct acylation of acetoacetate from acetoacetyl-CoA to the free thiol of the phosphopantetheine at the ACP active site (33) of holo -LovF. Using this direct approach, holo -LovF was incubated with 1mM acetoacetyl-CoA for 15, 60, 90 and 120 minutes prior to tryptic digestion and MS analysis (Figure 8A).…”

Section: Resultsmentioning

confidence: 99%

FT-ICR-MS Characterization of Intermediates in the Biosynthesis of the α-Methylbutyrate Side Chain of Lovastatin by the 277 kDa Polyketide Synthase LovF

Meehan

Xie²,

Zhao

et al. 2010

Biochemistry

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There are very few fungal polyketide synthases that have been characterized by mass spectrometry. In this paper we describe the in vitro reconstitution and FT-ICR-MS verification of the full activity of an intact 277 kDa fungal polyketide synthase LovF of the lovastatin biosynthetic pathway. We report here both the verification of the reconstitution of fully functional holo-LovF by using 13C-labelled malonyl-CoA to form α-methylbutyrate functionality, and also detection of five predicted intermediates covalently bound to the 4′-phosphopantetheine at the acyl carrier protein (ACP) active site utilizing the phosphopantetheine ejection assay and high resolution mass spectrometry. Under in vitro conditions, the diketide acetoacetyl-intermediate did not accumulate on the ACP active site of holo-LovF following incubation with malonyl-CoA substrate. We found that incubation of holo-LovF with acetoacetyl-CoA served as an effective means of loading the diketide intermediate onto the ACP active site of LovF. Our results, demonstrate that subsequent α-methylation of the acetoacetyl intermediate stabilizes the intermediate onto the ACP active site, and facilitates the formation and mass spectrometric detection of additional intermediates en route to the formation of α-methylbutyrate.

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Section: Resultsmentioning

confidence: 99%

FT-ICR-MS Characterization of Intermediates in the Biosynthesis of the α-Methylbutyrate Side Chain of Lovastatin by the 277 kDa Polyketide Synthase LovF

Meehan

Xie²,

Zhao

et al. 2010

Biochemistry

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“…4‐Propyloxetan‐2‐one (VM016) : Yield 42 % (48 mg); colorless oil; 1 H NMR (200 MHz, CDCl 3 ): δ =4.60–4.45 (m, 1 H), 3.52 (dd, J =16 Hz, 6 Hz, 1 H), 3.06 (dd, J =16 Hz, 6 Hz, 1 H), 2.00–1.65 (m, 2 H), 1.64–1.32 (m, 2 H), 0.99 ppm (t, J =7 Hz, 3 H); 13 C NMR (50 MHz, CDCl 3 ): δ =168.4, 71.1, 42.9, 36.7, 18.3, 13.7 ppm; HRMS m / z [ M +Na] + calcd for C 6 H 10 O 2 Na + : 137.0573, found: 137.0576; Anal. calcd for C 6 H 10 O 2 : C 63.14, H 8.83, found: C 62.97, H 8.92.…”

Section: Methodsmentioning

confidence: 99%

Synthesis of Long‐Chain β‐Lactones and Their Antibacterial Activities against Pathogenic Mycobacteria

et al. 2019

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In the quest for new antibacterial agents, a series of novel long‐ and medium‐chain mono‐ and disubstituted β‐lactones was developed. Their activity against three pathogenic mycobacteria—M. abscessus, M. marinum, and M. tuberculosis—was assessed by the resazurin microtiter assay (REMA). Among the 16 β‐lactones synthesized, only 3‐hexadecyloxetan‐2‐one (VM005) exhibited promising activity against M. abscessus, whereas most of the β‐lactones showed interesting activities against M. marinum, similar to that of the classical antibiotic, isoniazid. Regarding M. tuberculosis, six compounds were found to be active against this mycobacterium, with β‐lactone VM008 [trans‐(Z)‐3‐(hexadec‐7‐en‐1‐yl)‐4‐propyloxetan‐2‐one] being the best growth inhibitor. The promising antibacterial activities of the best compounds in this series suggest that these molecules may serve as leads for the development of much more efficient antimycobacterial agents.

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“…23, 24 Incorporation of an alkyne functionality at various ring positions provides a chemical handle for conjugation with fluorophores (Figure 2). The thioester-ACP resulting from incubation of holo -ACP with a propargyloxycarbonyl (Poc)-activated β-lactam was shown to be competitive with traditional N -acetylcysteamine (SNAc) thioesters for KS acylation.…”

Section: Introductionmentioning

confidence: 99%

A mechanism-based fluorescence transfer assay for examining ketosynthase selectivity

et al. 2012

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Since their discovery, polyketide synthases have received massive attention from researchers hoping to harness their potential as a platform for generating new and improved therapeutics. Despite significant strides toward this end, inherent specificites within the enzymes responsible for polyketide production have severely limited these efforts. We have developed a mechanism-based, fluorescence transfer assay for a key enzyme component of all polyketide synthases, the ketosynthase domain. As demonstrated, this method can be used with both ketosynthase-containing didomains and full modules. As proof of principle, the ketosynthase domain from module 6 of the 6-deoxyerythronolide synthase is examined for its ability to accept a variaty of simple thioester substrates. Consistent with its natural hexaketide substrate, we find that this ketosynthase prefers longer, α-branched thioesters and its ability to distinguish these structural features is quite remarkable. Substrate electronics are also tested via a variety of p-substituted aromatic groups. In all, we expect this technique to find considerable use in the field of polyketide biosynthesis and engineering due to its extraordinary simplicity and very distinct visible readout.

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Direct Acylation of Carrier Proteins with Functionalized β-Lactones

Cited by 15 publications

References 20 publications

FT-ICR-MS Characterization of Intermediates in the Biosynthesis of the α-Methylbutyrate Side Chain of Lovastatin by the 277 kDa Polyketide Synthase LovF

FT-ICR-MS Characterization of Intermediates in the Biosynthesis of the α-Methylbutyrate Side Chain of Lovastatin by the 277 kDa Polyketide Synthase LovF

Synthesis of Long‐Chain β‐Lactones and Their Antibacterial Activities against Pathogenic Mycobacteria

A mechanism-based fluorescence transfer assay for examining ketosynthase selectivity

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