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Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes
Published on: October 3, 2019
Selectivity, directionality, and promiscuity in peptide processing from a Bacillus sp. Al Hakam cyclodehydratase
Joel O Melby1, Kyle L Dunbar, Nhat Q Trinh
1Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
Journal of the American Chemical Society
|March 10, 2012
Summary
Thiazole/oxazole-modified microcins (TOMMs) are ribosomal natural products. This study reveals unique C- to N-terminal processing and remarkable substrate flexibility in TOMM biosynthesis, enabling novel combinatorial library design.
Area of Science:
- Biochemistry
- Natural Products Chemistry
- Synthetic Biology
Background:
- Thiazole/oxazole-modified microcins (TOMMs) are a class of ribosomal natural products.
- Their structures are derived from precursor peptides containing cysteine, serine, and threonine residues.
- Combinatorial biosynthesis offers a route to generate novel TOMM derivatives with altered structures and functions.
Purpose of the Study:
- To investigate the enzymatic promiscuity and substrate tolerance of a TOMM biosynthetic system from Bacillus sp. Al Hakam.
- To characterize the processing directionality and substrate flexibility of TOMM formation.
- To assess the potential for using unnatural substrates in designing artificial TOMMs.
Main Methods:
- Analysis of an uncharacterized TOMM gene cluster in Bacillus sp. Al Hakam.
- Fourier transform tandem mass spectrometry (FT-MS/MS) for structural elucidation.
- Assessment of cognate, noncognate, and unnatural precursor peptides.
Main Results:
- Azole ring formation was regio- and chemoselective.
- TOMM biosynthesis proceeded in a unique C- to N-terminal directionality.
- The biosynthetic machinery exhibited flexibility in the +1 position, accommodating charged amino acids and bisheterocyclization, with a modest bias for glycine at the -1 position.
- Unnatural substrates were processed predictably.
Conclusions:
- The TOMM biosynthetic machinery demonstrates significant substrate flexibility and tolerance, including towards unnatural substrates.
- Understanding this enzymatic promiscuity is crucial for designing combinatorial libraries to discover artificial TOMMs with desired biological activities.
- The unique C- to N-terminal processing mechanism expands the known diversity of ribosomal natural product biosynthesis.
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