Research
In the hunt for improved antibiotics, it is important to realize that natural products have been, without question, the most prolific source of all medicines. With the advent of massively parallel DNA sequencing, it has become apparent that our knowledge of natural product structure and function is astonishingly incomplete. Exploration of uncharted natural product chemical space will undoubtedly lead to improved, and entirely new, medicines. Thus, our group focuses on elucidating the biosynthesis, structure, and function of natural products. Our primary focus has been on thiazole/oxazole-containing peptides. Characterized examples have activities ranging from antibacterials to virulence-promoting toxins. Therefore, the study of these peptidic natural products allows us to not only better understand bacterial virulence (where pharmacological intervention would constitute a pathogen-specific approach to bacterial infection) but also explore unique chemical architectures, ideally positioning us to introduce new structural classes of antibiotics. Our recent successes and focal areas are highlighted below by research project.
YcaO chemistry
We are studying the structural and chemical nature of a unique class of biosynthetic enzymes.
Recent Publications
Liu, A.; Si, Y.; Dong, S.; Mahanta, N.; Penkala, H.N.; Nair, S.K.; Mitchell, D.A. “Functional elucidation of TfuA in peptide backbone thioamidation.” Nat. Chem. Biol. (2021). doi:https://doi.org/10.1038/s41589-021-00771-0
Nayak, D.D.; Liu, A.; Agrawal, N.; Rodriguez-Carerro, R.; Dong, S.H.; Mitchell, D.A.; Nair, S.K.; Metcalf, W.W. “Functional interactions between posttranslationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans.” PLoS Biol. (2020). doi:https://doi.org/10.1371/journal.pbio.3000507
Dong, S.H.; Liu, A.; Mahanta, N.; Mitchell, D.A., Nair, S.K. “Mechanistic basis for ribosomal peptide backbone modifications.” ACS Cent. Sci., 5: 842-851 (2019). doi:10.1021/acscentsci.9b00124
Mahanta, N.; Szantai-Kis, D.M.; Petersson, E.J.; Mitchell, D.A. “Biosynthesis and chemical applications of thioamides.” ACS Chem. Biol., 14: 142-163 (2019). doi:10.1021/acschembio.8b01022
The defining chemical feature of the linear azole-containing peptide (LAP) family of natural products are thiazol(in)e and (methyl)oxazol(in)e heterocycles, which derive from cysteine and serine/threonine residues of a ribosomally produced precursor peptide. These heterocycles also appear ubiquitously in the thiopeptides as well as in certain cyanobactins . Upon heterocyclization, an otherwise unstructured peptide becomes rigidified and able to elicit a biological function. It is common for numerous residues of the precursor peptide to undergo heterocyclization, among other posttranslational modifications, to yield the mature natural product. Our group investigated azoline/azole biosynthesis given that they are found in many pharmacologically active compounds and, prior to our work, the details of their formation were poorly understood.
In a two-step sequence, an ATP-dependent cyclodehydration first forms the azoline heterocycle, followed by an optional FMN-dependent dehydrogenation to afford the aromatic azole. (catalyzed by C/D) first forms the azoline heterocycle, followed by an optional FMN-dependent dehydrogenation (catalyzed by B) to afford the aromatic azole. Past attempts to gain deeper insights into the mechanism of heterocyclization and the specificity of the enzyme complex has been stymied by poor protein solubility, stability and difficulties monitoring heterocycle formation. Bioinformatically, we identified linear azol(in)e-containing peptide clusters that did not suffer from the same pitfalls, allowing us to answer previously intractable questions regarding the mechanistic details of linear azol(in)e-containing peptide biosynthesis. These investigations deciphered the regio- and chemoselectivity of substrate processing by a rather promiscuous cyclodehydratase and established the mechanistic role of ATP hydrolysis in the cyclodehydration reaction. This work revealed a new biological role for ATP, which is used to directly phosphorylate peptide backbone oxygens. Moreover, we have shown by NMR and other biochemical assays that it is actually the D-protein component, and not the C-protein, that performs the cyclodehydration reaction. The function of these collaborating proteins was heretofore not dissectible.
More recently we have been investigating divergent members of the enzyme family that includes the cyclodehydratase shown above. Collectively, these proteins are referred to as
YcaO enzymes but only about involved in azoline biosynthesis. Genome neighborhood analysis predicts that out of all currently identified YcaO proteins (n=14,000) less than one-third are encoded in a context that would allow for azoline formation. Approximately 10% of the total are encoded next to a gene named tfuA, but the predicted precursor peptide often lack the plethora of beta-nucleophile containing residues required for heterocycle formation. We have found that these biosynthetic gene clusters are responsible for peptidic thioamidation and require sulfur from an outside source. Interestingly, methanogenic archaea always encode a YcaO protein. We have recently found that the YcaO is directly responsible for the
thioamidation of methyl-coenzyme M reductase, one of the most important proteins in the global carbon cycle. YcaO proteins are also responsible for the formation of the macrolactamidine functional group found in bottromycin. In this example, the N-terminus of the peptide, as opposed to an adjacent Cys/Ser/Thr or exogenous sulfide, is the nucleophile that attacks the activate amide backbone. Nature clearly has employed YcaO proteins widely to perform chemistry on the backbone of peptides and we anticipate new reactions to be discovered within and beyond this enzyme family.
Lasso Peptides
We bioinformatically mapped lasso peptide clusters and structures on a large scale and are using this information to study these antibacterial peptides.
Recent Publications
Kretsch, A.M.; Gadgil, M.G.; DiCaprio, A.J.; Barrett, S.E.; Kille, B.L.; Si, Y.; Zhu, L.; Mitchell, D.A. “Peptidase activation by a leader peptide-Bound RiPP recognition element.” Biochemistry, 62 956-967 (2023). doi: 10.1021/acs.biochem.2c00700
Si, Y.; Kretsch, A.M.; Daigh, L.M.; Burk, M.J.; Mitchell, D.A. “Cell-Free biosynthesis to evaluate lasso peptide formation and enzyme–substrate tolerance.” J. Am. Chem. Soc., 143: 5917–5927 (2021). doi:10.1021/jacs.1c01452
Harris, L.A.; Saint-Vincent, P.M.B.; Guo, X.R.; Hudson, G.A.; DiCaprio, A.J.; Zhu, L.; Mitchell, D.A. “Reactivity-Based Screening for Citrulline-Containing Natural Products Reveals a Family of Bacterial Peptidyl Arginine Deiminases.” ACS Chem. Biol., 15: 3167–3175 (2020). doi:10.1021/acschembio.0c00685
Lasso peptides have a variety of characterized bioactivity and hold great promise as a therapeutic scaffold due to their exemplary stability to proteases and extreme environments.
To facilitate their discovery from natural sources, we developed Rapid ORF Description and Evaluation Online (RODEO) which automates the time-consuming process of annotating biosynthetic gene clusters and predicting likely lasso peptide precursors. Using RODEO, we have predicted thousands of lasso peptide gene clusters present in GenBank, the most comprehensive list to date. With this set of all lasso clusters and precursor peptides, we are poised to expand what is known regarding this expansive natural product family. We aim to discover and characterize lasso peptides with novel post-translational modifications, structures, and bioactivity.
Despite our ability to accurately predict lasso peptide gene clusters, we understand little about the enzymology of the core biosynthetic enzymes. In particular, many questions remain about how the cyclase facilitates lasso peptide folding and ties the lasso knot.
In 2019, RODEO enabled the discovery and characterization of the lasso peptide Fusilassin. This thermophilic derived lasso peptide system is only the second to date compatible with in vitro experiments, opening the doors for in-depth biochemical characterization of its enigmatic biosynthetic enzymes. Using cell-free technology, we were able to show that Fusilassin’s biosynthetic enzymes are extremely promiscuous, tolerating approximately 2 million diverse ring sequences. Combining the vast natural diversity of lasso peptides with their cyclase’s promiscuous substrate tolerance will enable the creation of customized, unnatural lasso peptides with interesting biological activity.
Thiopeptides
We are studying the biosynthesis of thiopeptide natural products.
Recent Publications
Nguyen, D.T.; Le, T.T.; Rice, A.J.; Hudson, G.A.; van der Donk, W.A.; Mitchell, D.A. “Accessing Diverse Pyridine-Based Macrocyclic Peptides by a Two-Site Recognition Pathway” J. Am. Chem. Soc. (2022). doi:https://doi.org/10.1021/jacs.2c02824
Hudson, G.A.; Hooper, A.R.; DiCaprio, A.J.; Sarlah, D.; Mitchell, D.A. “Structure prediction and synthesis of pyridine-based macrocyclic peptide natural products.” Org. Lett. (2020). doi:https://doi.org/10.1021/acs.orglett.0c02699
Schwalen, C.J., Hudson, G.A., et al. “Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics.” J. Am. Chem. Soc. (2018)
doi:10.1021/jacs.8b03896
Cogan, D.P., Hudson, G.A., et al. “Structural Insights into Enzymatic [4+2]-Aza-cycloaddition in Thiopeptide Antibiotic Biosynthesis.” Proc. Natl. Acad. Sci. (2017)
doi:10.1073/pnas.1716035114
The chemical hallmark of the linear azole-containing peptide (LAP) family of natural products are thiazol(in)e and (methyl)oxazol(in)e heterocycles installed by cyclodehydratases from cysteine and serine/threonine residues, respectively. These post-translational modifications impart rigidity on a peptidic natural product and in many cases are crucial for downstream enzymatic processing and bioactivity. Reported in a series of papers from 2012-2015, we thoroughly investigated the details of cyclodehydration, including the mechanistic enzymology of azoline biosynthesis, which uses ATP to directly activate the peptidic amide backbone, the structural details of the cyclodehydratase, revealing a novel ATP binding motif, and the orchestration of the flavin-dependent dehydrogenase which oxidizes azolines to corresponding azoles.
Further research revealed a new class of cyclodehydratases which, upon characterizing, deciphered how the peptidic substrate is recruited to the cyclodehydratase at the molecular level. Gaining a deeper understanding of cyclodehydration has enabled the study of more complicated systems, including the thiopeptides, a family of peptidic natural products with potent antibiotic activity towards a variety of drug-resistant bacteria. Despite their architectural complexity, thiopeptide biosynthesis proceeds through a LAP-like intermediate, and as such, feature cyclodehydratases and dehydrogenases for thiazole biosynthesis, but also feature lanthipeptide-like dehydratases and a [4+2] cycloaddition enzyme responsible for pyridine formation. Building upon our previous work on cyclodehydratases, and in collaboration with lanthipeptide biosynthetic experts in the laboratory of Prof. Wilfred van der Donk, we have reconstituted the core biosynthesis of thiomuracin, enabling production of these complicated molecules in vitro using only six enzymes on a precursor peptide substrate. Reconstitution also provided unprecedented insight into the precise details of thiopeptide biosynthesis, including the largely uncharacterized [4+2] cycloaddition enzyme. Further endeavors have yielded crucial insight into the timing and substrate specificity of thiomuracin biosynthesis and will provide a groundwork for enzymatically generating new thiopeptide analogs with desirable pharmacological properties.