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Biochemistry. 2019 Feb 26;58(8):1131-1140. doi: 10.1021/acs.biochem.8b01120. Epub 2019 Feb 13.

Characterization of a Hybrid Nonribosomal Peptide-Carbohydrate Biosynthetic Pathway in Photorhabdus luminescens.

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Department of Chemistry , Yale University , New Haven , Connecticut 06520 , United States.
Chemical Biology Institute , Yale University , West Haven , Connecticut 06516 , United States.
Department of Microbial Pathogenesis , Yale School of Medicine , New Haven , Connecticut 06536 , United States.


Advances in genome sequencing and analysis have afforded a trove of "orphan" bacterial biosynthetic pathways, many of which contain hypothetical proteins. Given the potential for these hypothetical proteins to carry out novel chemistry, orphan pathways serve as a rich reservoir for the discovery of new enzymes responsible for the production of metabolites with both fascinating chemistries and biological functions. We previously identified a rare hybrid nonribosomal peptide synthetase (NRPS)-carbohydrate genomic island in the entomopathogen Photorhabdus luminescens. Heterologous expression of the pathway led to the characterization of oligosaccharides harboring a 1,6-anhydro-β-d- N-acetyl-glucosamine moiety, but these new metabolites lacked modification by the NRPS machinery. Here, through the application of top-down protein mass spectrometry, pathway-targeted molecular networking, stable isotope labeling, and in vitro protein biochemistry, we complete the characterization of this biosynthetic pathway and identify the hybrid product of the pathway, a new "glycoamino acid" metabolite termed photolose. Intriguingly, a hypothetical protein served as a bridge to condense a glycyl unit derived from the NRPS machinery onto the free 1,6-anhydro-β-d- N-acetyl-glucosamine core. We further demonstrate that the gene cluster confers a growth advantage to antimicrobial peptide challenge.

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