Dataset Information

Identification of a Thioesterase Bottleneck in the Pikromycin Pathway through Full-Module Processing of Unnatural Pentaketides.

ABSTRACT: Polyketide biosynthetic pathways have been engineered to generate natural product analogs for over two decades. However, manipulation of modular type I polyketide synthases (PKSs) to make unnatural metabolites commonly results in attenuated yields or entirely inactive pathways, and the mechanistic basis for compromised production is rarely elucidated since rate-limiting or inactive domain(s) remain unidentified. Accordingly, we synthesized and assayed a series of modified pikromycin (Pik) pentaketides that mimic early pathway engineering to probe the substrate tolerance of the PikAIII-TE module in vitro. Truncated pentaketides were processed with varying efficiencies to corresponding macrolactones, while pentaketides with epimerized chiral centers were poorly processed by PikAIII-TE and failed to generate 12-membered ring products. Isolation and identification of extended but prematurely offloaded shunt products suggested that the Pik thioesterase (TE) domain has limited substrate flexibility and functions as a gatekeeper in the processing of unnatural substrates. Synthesis of an analogous hexaketide with an epimerized nucleophilic hydroxyl group allowed for direct evaluation of the substrate stereoselectivity of the excised TE domain. The epimerized hexaketide failed to undergo cyclization and was exclusively hydrolyzed, confirming the TE domain as a key catalytic bottleneck. In an accompanying paper , we engineer the standalone Pik thioesterase to yield a thioesterase (TES148C) and module (PikAIII-TES148C) that display gain-of-function processing of substrates with inverted hydroxyl groups.

SUBMITTER: Hansen DA

PROVIDER: S-EPMC5617803 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Identification of a Thioesterase Bottleneck in the Pikromycin Pathway through Full-Module Processing of Unnatural Pentaketides.

Hansen Douglas A DA Koch Aaron A AA Sherman David H DH

Journal of the American Chemical Society 20170919 38

Polyketide biosynthetic pathways have been engineered to generate natural product analogs for over two decades. However, manipulation of modular type I polyketide synthases (PKSs) to make unnatural metabolites commonly results in attenuated yields or entirely inactive pathways, and the mechanistic basis for compromised production is rarely elucidated since rate-limiting or inactive domain(s) remain unidentified. Accordingly, we synthesized and assayed a series of modified pikromycin (Pik) pentak ...[more]

PMID: 28836772

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Project description:Background:In the classical psychological refractory period (PRP) paradigm, two stimuli are presented in brief succession, and participants are asked to make separate speeded responses to both stimuli. Due to a central cognitive bottleneck, responses to the second stimulus are delayed, especially at short stimulus-onset asynchrony (SOA) between the two stimuli. Although the mechanisms of dual-task interference in the classical PRP paradigm have been extensively investigated, specific mechanisms underlying the cross-modal PRP paradigm are not well understood. In particular, it remains unknown whether the dominance of vision over audition manifests in the cross-modal PRP tasks. The present study aimed to investigate whether the visual dominance effect manifests in the cross-modal PRP paradigm. Methods:We adapted the classical PRP paradigm by manipulating the order of a visual and an auditory task: the visual task could either precede the auditory task or vice versa, at either short or long SOAs. Twenty-five healthy participants took part in Experiment 1, and thirty-three new participants took part in Experiment 2. Reaction time and accuracy data were calculated and further analyzed by repeated-measures analysis of variance. Results:The results showed that visual precedence in the Visual-Auditory condition caused larger impairments to the subsequent auditory processing than vice versa in the Auditory-Visual condition: a larger delay of second response was revealed in the Visual-Auditory condition (135 ± 10 ms) than the Auditory-Visual condition (88 ± 9 ms). This effect was found only at the short SOAs under the existence of the central bottleneck, but not at the long SOAs. Moreover, this effect occurred both when the single visual and the single auditory task were of equal difficulty in Experiment 1 and when the single auditory task was more difficult than the single visual task in Experiment 2. Conclusion:Results of the two experiments suggested that the visual dominance effect occurred under the central bottleneck of cognitive processing.

Dataset Information

Identification of a Thioesterase Bottleneck in the Pikromycin Pathway through Full-Module Processing of Unnatural Pentaketides.

Publications

Identification of a Thioesterase Bottleneck in the Pikromycin Pathway through Full-Module Processing of Unnatural Pentaketides.

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