Nonribosomal Peptide Syntheses (NRPS) Proteins and the Microbiota

The microbiota is a collective term used to describe all bacteria, fungi, viruses and archaea that reside in and on a human host. Most of these upwards of 5,000 different species of microorganisms work symbiotically with the host. Klebsiella oxytoca is an opportunistic bacteria found in ~10% of healthy individuals’ microbiota. Upon treatment with penicillin-based antibiotics, constitutive beta-lactamase enzymes allow K. oxytoca to not only survive, but thrive. This leads to dysbiosis and severe colon damage through the secondary metabolite tilimycin.

Nonribosomal peptide synthetases (NRPSs) synthesize complex peptide natural products such as tilivalline. These modular enzymes contain three core domains (adenylation, thiolation, and condensation, A). Using our knowledge of adenylation inhibitors, we have developed and characterized mimics targeting the adenylation domain in the tilimycin biosynthetic pathway (B-E). We believe biochemical examination of these domains and how they interact with each other may lend to optimization of inhibitors to block virulence factors while being nonmicrobicidal, minimizing off target effects on the delicate microbiota.

• Alexander EM, Kreitler DF, Guidolin V, Hurben AK, Drake E, Villalta PW, Balbo S, Gulick AM, Aldrich CC. Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella Oxytoca. ACS Infectious Diseases. 2020. [PDF]

• Miller, B. R., Drake, E. J., Shi, C., Aldrich, C. C., & Gulick, A. M. Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture. The Journal of Biological Chemistry. 2016. [PDF]

Pyridoxal-5-phosphate-dependent enzymes (PLP-DEs) catalyze an extraordinary diversity of chemical reactions in both primary and secondary metabolic pathways.  A number of FDA-approved drugs are known that covalently modify PLP-DEs exemplified by vigabatrin, carbidopa, D-cycloserine, and eflornithine used to treat epilepsy, Parkinson’s, tuberculosis, and African sleeping sickness, respectively. In general, many of these aforementioned drugs are characterized by substantial side effects that are incompletely understood, but likely due to inhibition of other functionally related enzymes. Activity-based protein profiling (ABPP) is a powerful technique that has become popular in the field of chemical biology for characterizing enzyme selectivity and target identification of tool compounds (A). We have successfully identified a probe capable of covalently labeling PLP-DEs (B), and seek to employ this probe in ABPP studies to identify and characterize PLP-DE reactivity.

• Brody SI, Buonomo JA, Orimoloye MO, Jia Z, Sharma S, Brown CD, Baughn AD, Aldrich CC. A Nucleophilic Activity-Based Probe Enables Profiling of PLP-Dependent Enzymes. Chembiochem. 2023 [PDF]