Megaenzyme Engineering

NRPS

Nonribosomal peptide synthetases (NRPS) are mega-enzyme complexes that produce a wide range of biologically active natural compounds, constituting an important source of useful therapeutics. They consist of multiple modules, which act in assembly-line fashion to incorporate the amino acid building blocks into the growing peptide chain. Each module contains several core domains that catalyze the individual reaction steps needed for amino acid activation, loading onto the enzyme, and peptide bond formation. The peptidyl product can be further diversified by inclusion of additional domains, for example for epimerization, cyclization, or oxidation/reduction. To enable repurposing NRPS domains for novel substrates, we have developed a toolbox of high-throughput methods to engineer individual domains. Efficient incorporation of “clickable” and β-amino acids into nonribosomal peptides illustrates the potential of such approaches for studying and engineering NRPS assembly lines for the production of novel tailored peptides.

Cyanophycin synthetase, another remarkable multi-functional macromolecular machine, is responsible for the biosynthesis of cyanophycin, a nitrogen- and carbon-storage polymer found in many cyanobacteria and some heterotrophic bacteria. Structural and biochemical studies, in collaboration with Martin Schmeing (McGill University, Montreal), have revealed how this enzyme catalyzes non-ribosomal peptide polymerization by sequentially activating aspartate residues and conjugating arginine side chains in a process that requires Mg2+, ATP, and a short peptide primer.

Selected Publications

D. Hilvert & T.M. Schmeing. High-throughput engineering of biosynthetic assembly lines. Nat. Chem. Biol. 20, 671–672 (2024)

I.B. Folger, N.F. Frota, A. Pistofidis, D.A. Hansem, D.L. Niquille, T.M. Schmeing & D. Hilvert. High-throughput reprogramming of an NRPS condensation domain. Nat. Chem. Biol. 20, 761–769 (2024)

A. Camus, M. Gantz, D. Hilvert. High-throughput engineering of nonribosomal extension modules. ACS Chem. Biol. 18, 2516–2523 (2023)

I. Sharon, D. Hilvert & T.M. Schmeing. Cyanophycin and its biosynthesis: Not hot but very cool. Nat. Prod. Rep. 40, 1479-1497 (2023)

L.M.D. Markus, I. Sharon, K. Munro, M. Grogg, D. Hilvert, M. Strauss, T.M. Schmeing. Structure and function of a hexameric cyanophycin synthetase 2. Protein Sci. 32, e4685 (2023)

A. Camus, G. Truong, P. Mittl, G. Markert & D. Hilvert. Reprogramming nonribosomal peptide synthetases for site-specific insertion of α-hydroxy acids. J. Am. Chem. Soc. 144, 17567–17575 (2022)

I. Sharon, M. Grogg, D. Hilvert & T.M. Schmeing. The structure of cyanophycinase in complex with a cyanophycin degradation intermediate. Biochim. Biophys. Acta Gen. Subj. 1866, 130217 (2022)

I. Sharon, S. Pinus, M. Grogg, N. Moitessier, D. Hilvert & T.M. Schmeing. A cryptic third active site in cyanophycin synthetase creates primers for polymerization. Nat. Commun. 13, 3923 (2022)

I. Sharon, M. Grogg, D. Hilvert & T.M. Schmeing. Structure and function of the β-Asp-Arg polymerase cyanophycin synthetase 2. ACS Chem. Biol. 17, 670–679 (2022)

I. Sharon, A.S. Haque, M. Grogg, I. Lahiri, D. Seebach, A.E. Leschziner, D. Hilvert & T.M. Schmeing. Structure and function of the amino acid polymerase cyanophycin synthetase. Nat. Chem. Biol. 17, 1101-1110 (2021)

D.L. Niquille, I.B. Folger, S. Basler & D. Hilvert. Biosynthetic functionalization of nonribosomal peptides. J. Am. Chem. Soc. 143, 2736–2740 (2021)

D.L. Niquille, D.A. Hansen & D. Hilvert. Reprogramming nonribosomal peptide synthesis by surgical mutation. Syn. Lett. 30, 2123-2130 (2019)

D.L. Niquille, D.A. Hansen, T. Mori, D. Fercher, H. Kries & D. Hilvert. Nonribosomal biosynthesis of backbone-modified peptides. Nat. Chem. 10, 282-287 (2018)

H. Kries, D.L. Niquille & D. Hilvert. A subdomain swap strategy for reengineering nonribosomal peptides. Chem. Biol. 22, 640–648 (2015)

H. Kries, R. Wachtel, A. Pabst, B. Wanner, D. Niquille & D. Hilvert. Reprogramming nonribosomal peptide synthetases for “clickable” amino acids. Angew. Chem. Int. Ed. 53, 10105-10108 (2014)