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Selectivity, directionality, and promiscuity in peptide processing from a Bacillus sp. Al Hakam cyclodehydratase.

ABSTRACT: The thiazole/oxazole-modified microcins (TOMMs) represent a burgeoning class of ribosomal natural products decorated with thiazoles and (methyl)oxazoles originating from cysteines, serines, and threonines. The ribosomal nature of TOMMs allows for the generation of derivative products from mutations in the amino acid sequence of the precursor peptide, which ultimately manifest in differing structures and, sometimes, biological functions. Employing a TOMM system for the purpose of creating new structures and functions via combinatorial biosynthesis requires processing machinery that can tolerate highly variable substrates. In this study, TOMM enzymatic promiscuity was assessed using a currently uncharacterized cluster in Bacillus sp. Al Hakam. As determined by Fourier transform tandem mass spectrometry (FT-MS/MS), azole rings were formed in both a regio- and chemoselective fashion. Cognate and noncognate precursor peptides were modified in an overall C- to N-terminal directionality, which to date is unique among characterized ribosomal natural products. Studies focused on the inherent promiscuity of the biosynthetic machinery elucidated a modest bias for glycine at the preceding (-1) position and a remarkable flexibility in the following (+1) position, even allowing for the incorporation of charged amino acids and bisheterocyclization. Two unnatural substrates were utilized as the conclusive test of substrate flexibility, of which both were processed in a predictable fashion. A greater understanding of substrate processing and enzymatic tolerance toward unnatural substrates will prove beneficial when designing combinatorial libraries to screen for artificial TOMMs that exhibit desired activities.

SUBMITTER: Melby JO

PROVIDER: S-EPMC3324979 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Selectivity, directionality, and promiscuity in peptide processing from a Bacillus sp. Al Hakam cyclodehydratase.

Melby Joel O JO Dunbar Kyle L KL Trinh Nhat Q NQ Mitchell Douglas A DA

Journal of the American Chemical Society 20120308 11

The thiazole/oxazole-modified microcins (TOMMs) represent a burgeoning class of ribosomal natural products decorated with thiazoles and (methyl)oxazoles originating from cysteines, serines, and threonines. The ribosomal nature of TOMMs allows for the generation of derivative products from mutations in the amino acid sequence of the precursor peptide, which ultimately manifest in differing structures and, sometimes, biological functions. Employing a TOMM system for the purpose of creating new str ...[more]

PMID: 22401305

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Project description:During the continued isolation of different bacteria from highly diverse, low human activity environments in Ghana and the subsequent characterization and biological activity studies of their secondary metabolites, we found both Gram-positive and Gram-negative Bacillus strains to be ubiquitous and widespread. One of such strains, the Ghanaian novel Bacillus sp. strain DE2B was isolated from rhizosphere soils collected from the Digya National Park in Ghana. Chromatographic purifications of the fermented culture extract of the strain DE2B, led to the isolation of a cyclic lipopeptide, digyalipopeptide A (1). Using 1D and 2D NMR data, mass spectrometry sequence tagging, advanced Marfey's analysis, and the GNPS molecular networking we solved the full structure of digyalipopeptide A (1). We found that compound 1 is a member of a somewhat homologous series of peptides produced as a mixture by the strain containing the same amino acid sequence in the cyclic peptide backbone but differing only by the length of aliphatic fatty acid side chains. When tested against Trypanosoma brucei subsp. brucei strain GUTat 3.1 and Leishmania donovani (Laveran and Mesnil) Ross (D10), digyalipopeptide A (1) gave IC50 values of 12.89 µM (suramin IC50 0.96 µM) and 4.85 µM (amphotericin B IC50 4.87 µM), respectively. Furthermore, digyalipopeptide A (1) produced IC50 values of 10.07 µM (ampicillin IC50 0.18 µM) and 10.01 µM (ampicillin IC50 1.53 µM) for Staphylococcus aureus and Shigella sonnei, respectively. The selectivity and toxicity profile of compound 1 was investigated using normal cell lines, macrophages RAW 264.7. When tested against normal macrophages, compound 1 gave an IC50 value of 71.32 μM. Selectivity indices (SI) were obtained by calculating the ratio of the IC50 in RAW 264.7 to the IC50 in the respective microbe and neglected parasite. In the presence of RAW 264.7 cell lines, compound 1 was particularly selective towards Leishmania donovani (Laveran and Mesnil) Ross (D10) with an SI value of 14.71. The bioactivity studies conducted confirm the role of these cyclic lipopeptides as defense chemicals in their natural environment and their ability to be biologically active across different species.

Project description:ObjectiveChildren with ADHD demonstrate impaired performance on a wide range of neuropsychological tests. It is unclear, however, whether ADHD is associated with many neurocognitive deficits or whether a small number of impairment(s) broadly influence test performance. The current study tests competing model predictions regarding two candidate causal mechanisms in ADHD: information processing speed and working memory.MethodA well-characterized sample of 86 children (Mage = 10.52, SDage = 1.54; 34 girls; 64% Caucasian/Non-Hispanic) with ADHD (n = 45) and without ADHD (n = 41) completed eight fully crossed experimental tasks that systematically manipulated working memory (BF?? = 1.80 × 10?³) and information processing speed (drift rate; BF?? = 7.61 × 10?).ResultsBayesian mixed-model ANOVAs indicated that increasing working memory demands produced significant reductions in information processing speed (drift rate; BF?? = 5.82 × 10??). In contrast, experimentally reducing children's information processing speed did not significantly change their working memory performance (BF?? = 1.31). ADHD status interacted with the working memory manipulation, such that the ADHD and non-ADHD groups showed equivalently high accuracy under the encoding-only conditions (BF?? = 3.45) but differed significantly under high working memory conditions (encoding + recall; BF?? = 19.58). Importantly, however, ADHD status failed to interact with (a) the working memory manipulation to differentially affect information processing speed and (b) the information processing speed manipulation to differentially affect working memory performance (all BF?? > 4.25).ConclusionsThese findings indicate that top-down executive control exerts significant effects on children's ability to quickly process information, but that working memory deficits and slowed information processing speed appear to be relatively independent impairments in ADHD. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

Dataset Information

Selectivity, directionality, and promiscuity in peptide processing from a Bacillus sp. Al Hakam cyclodehydratase.

Publications

Selectivity, directionality, and promiscuity in peptide processing from a Bacillus sp. Al Hakam cyclodehydratase.

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