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Engineering unnatural variants of plantazolicin through codon reprogramming.

ABSTRACT: Plantazolicin (PZN) is a polyheterocyclic natural product derived from a ribosomal peptide that harbors remarkable antibiotic selectivity for the causative agent of anthrax, Bacillus anthracis. To simultaneously establish the structure-activity relationship of PZN and the substrate tolerance of the biosynthetic pathway, an Escherichia coli expression strain was engineered to heterologously produce PZN analogues. Variant PZN precursor genes were produced by site-directed mutagenesis and later screened by mass spectrometry to assess post-translational modification and export by E. coli. From a screen of 72 precursor peptides, 29 PZN variants were detected. This analogue collection provided insight into the selectivity of the post-translational modifying enzymes and established the boundaries of the natural biosynthetic pathway. Unlike other studied thiazole/oxazole-modified microcins, the biosynthetic machinery appeared to be finely tuned toward the production of PZN, such that the cognate enzymes did not process even other naturally occurring sequences from similar biosynthetic clusters. The modifying enzymes were exquisitely selective, installing heterocycles only at predefined positions within the precursor peptides while leaving neighboring residues unmodified. Nearly all substitutions at positions normally harboring heterocycles prevented maturation of a PZN variant, though some exceptions were successfully produced lacking a heterocycle at the penultimate residue. No variants containing additional heterocycles were detected, although several peptide sequences yielded multiple PZN variants as a result of varying oxidation states of select residues. Eleven PZN variants were produced in sufficient quantity to facilitate purification and assessment of their antibacterial activity, providing insight into the structure-activity relationship of PZN.

SUBMITTER: Deane CD

PROVIDER: S-EPMC3783559 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Engineering unnatural variants of plantazolicin through codon reprogramming.

Deane Caitlin D CD Melby Joel O JO Molohon Katie J KJ Susarrey Aziz R AR Mitchell Douglas A DA

ACS chemical biology 20130703 9

Plantazolicin (PZN) is a polyheterocyclic natural product derived from a ribosomal peptide that harbors remarkable antibiotic selectivity for the causative agent of anthrax, Bacillus anthracis. To simultaneously establish the structure-activity relationship of PZN and the substrate tolerance of the biosynthetic pathway, an Escherichia coli expression strain was engineered to heterologously produce PZN analogues. Variant PZN precursor genes were produced by site-directed mutagenesis and later scr ...[more]

PMID: 23823732

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Project description:ImportanceLife expectancy is decreasing in the US. Without national efforts to address factors that support policies and programs directed at children living in areas of concentrated poverty, life expectancy will likely continue to decline while costs and suffering associated with unnatural deaths will increase.ObjectiveTo identify which childhood factors are associated with death from unnatural causes through midadulthood.Design, setting, and participantsFor this cohort study, longitudinal data on childhood characteristics came from a group-randomized intervention trial implemented in Baltimore City Public Schools, Baltimore, Maryland (baseline 1985-1986; all students entering first grade were selected to participate at age 6 years). Participants were followed up to midadulthood with a National Death Index search through December 31, 2020. Data analysis was performed from February to May 2023.ExposuresExposures included individual factors (ie, sociodemographic characteristics, teacher-reported aggressive behavior, self-reported depression, anxiety, early alcohol and cannabis use, and assaultive violence exposure), family and peer factors (ie, household structure and education level, deviant peer affiliation, and parental monitoring), and neighborhood factors (ie, rates of neighborhood assault and public assistance).Main outcomes and measuresThe main outcome was unnatural death, defined as death due to unintentional injury, suicide, and homicide. A National Death Index search ascertained participants who died by age 41 to 42 years and cause of death. Multivariable Cox proportional hazards models were used to identify whether the exposures were independently associated with future mortality by unnatural causes.ResultsThe initial trial included 2311 children, and longitudinal data were available for 2180 participants (median [IQR] age in first grade, 6.3 [6.0-6.5] years; 1090 female [50.0%]; 1461 Black [67.0%]; 1168 received free or reduced lunch in first grade [53.6%]). A total of 111 male participants (10.2%) and 29 female participants (2.7%) died; among those who died, 96 male participants (86.5%) and 14 female participants (48.3%) died of unnatural causes. Two factors remained significantly associated with mortality from unnatural causes: female sex was associated with reduced risk (hazard ratio, 0.13; 95% CI, 0.08-0.22), and neighborhood public assistance was associated with increased risk (hazard ratio, 1.89; 95% CI, 1.09-3.30).Conclusions and relevanceIn this urban population-based cohort study, no modifiable risk factors of mortality at the level of the individual (eg, depression or anxiety and substance use) or the family (eg, household education level) were identified. However, the degree of neighborhood poverty in early childhood was significantly associated with death by unnatural causes in early adulthood, suggesting that economic policies are needed to advance health equity in relation to premature mortality.

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Engineering unnatural variants of plantazolicin through codon reprogramming.

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Engineering unnatural variants of plantazolicin through codon reprogramming.

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