Discovery and application of new enzymes for amino acid and peptide modification

/Abstract The discovery of new bioactive compounds used for human medicine is driven by our ability to rapidly and efficiently generate new chemical structures. One major challenge in this aim is the development of synthetic strategies that allow us to prepare molecules with the three-dimensional structural complexity that is needed for selective recognition of cellular targets. Along these lines, it has been shown that small molecule natural products, peptides, and proteins all possess the structural features needed to efficiently and successfully recognize macromolecules of interest but remain difficult to produce on scale through traditional synthetic chemistry. The long-term goals of this research program are to develop new biocatalytic tools in the form of enzymes and metabolic pathways to address this challenge. This proposal focuses on the development of methods to discover and engineer enzymes and pathways involved in the modification of amino acids and peptides. Amino acids serve as key chiral building blocks for the cell, producing target molecules ranging from functional non- canonical amino acids, alkaloid natural products, bioactive peptides, and proteins. Only certain classes of these products, such as those made by non-ribosomal peptide synthases or the ribosome, can be readily identified through established bioinformatic methods. Our lab has defined several classes of privileged protein families involved in the transformation of amino acids and propose to discover, characterize, and engineer these new enzymes as synthetic tools for the preparation of targets ranging from small-molecule nitrogen heterocycles to modified peptides and to protein conjugates. In the proposed work, we focus on non-heme Fe(II)/α-ketoglutarate dependent enzymes including halogenases, hydroxylases, and other transformations that carry out the C-H activation of inert C(sp3)-sites to introduce synthetically useful functional groups such as Cl, Br, N3, or keto groups for downstream reactions. We also explore the enzymology of the ATP-Grasp family of enzymes, which form amide linkages between amino acids or peptides and various small molecules. Amide bonds remain essential for medicinal chemistry and we seek to develop ATP- Grasp enzymes both for solution-phase approaches for peptide synthesis as well as biocatalytic site-selective protein modification. Project Number: 1R35GM161243-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: MICHELLE CHANG | Institution: PRINCETON UNIVERSITY, Princeton, NJ | Award Amount: $451,000 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award - E Study Section[MRAE] View on NIH RePORTER: https://reporter.nih.gov/project-details/11259231