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Abstract Beta-hydroxy non-standard amino acids (β-OH-nsAAs) have utility as small molecule drugs, precursors for beta-lactone antibiotics, and building blocks for polypeptides. While the L-threonine transaldolase (TTA), ObiH, is a promising enzyme for β-OH-nsAA biosynthesis, little is known about other natural TTA sequences. We ascertained the specificity of the TTA enzyme class more comprehensively by characterizing 12 candidate TTA gene products across a wide range (20-80%) of sequence identities. We found that addition of a solubility tag substantially enhanced the soluble protein expression level within this difficult-to-express enzyme family. Using an optimized coupled enzyme assay, we identified six TTAs, including one with less than 30% sequence identity to ObiH that exhibits broader substrate scope, two-fold higher L-Threonine (L-Thr) affinity, and five-fold faster initial reaction rates under conditions tested. We harnessed these TTAs for first-time bioproduction of β-OH-nsAAs with handles for bio-orthogonal conjugation from supplemented precursors during aerobic fermentation of engineeredEscherichia coli, where we observed that higher affinity of the TTA for L-Thr increased titer. Overall, our work reveals an unexpectedly high level of sequence diversity and broad substrate specificity in an enzyme family whose members play key roles in the biosynthesis of therapeutic natural products that could benefit from chemical diversification.
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Quantitative chemoproteomic profiling reveals multiple target interactions of spongiolactone derivatives in leukemia cells
The spongiolactones are marine natural products with an unusual rearranged spongiane skeleton and a fused β-lactone ring. These compounds have potential anticancer properties but their mode of action has yet to be explored. Here we employ activity-based protein profiling to identify the targets of a more potent spongiolactone derivative in live cancer cells, and compare these to the targets of a simpler β-lactone. These hits provide the first insights into the covalent mechanism of action of this natural product class.
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- Award ID(s):
- 1546973
- PAR ID:
- 10100468
- Date Published:
- Journal Name:
- Chemical Communications
- Volume:
- 53
- Issue:
- 95
- ISSN:
- 1359-7345
- Page Range / eLocation ID:
- 12818 to 12821
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/C7CC04990K
