CURE-ating the Substrate Scope and Functional Residues of Nonheme Iron(II) α-Ketoglutarate-Dependent Hydroxylase BesE

Nonheme iron(II) α-ketoglutarate-dependent dioxygenases (Fe/αKGs) play important roles in functionalizing biological substrates from individual amino acids to macromolecules. BesE, a unique homologue from the actinobacterial β-ethynylserine biosynthetic pathway, catalyzes a highly selective hydroxylation on dipeptide substrate γ-l-glutamyl-l-propargylglycine (1). Inspired by this transformation, our year-long Course-based Undergraduate Research Experience (CURE) laboratory interrogated BesE catalysis using an interdisciplinary approach. After establishing a modular chemical synthesis of 1, we rationally designed 14 non-native analogues with key alterations to specific substrate moieties putatively involved in enzymatic recognition. Following in vitro enzymology and the application of chemical derivatization techniques compatible with all substrate analogues and putative products, we determined that the terminal alkyne moiety is not essential while reinforcing the significance of the γ-glutamyl moiety for hydroxylation activity. Thirteen rationally designed BesE mutants established the importance of polar active site residues for substrate recognition and catalysis. This work establishes a baseline of BesE recognition from both a chemical and biochemical perspective and contributes to the growing understanding of Fe/αKG recognition on biomedically relevant targets. Moreover, this contributes to the growing examples of using natural products and their biosynthetic enzymology as a vibrant platform for the interdisciplinary training of early career biomedical researchers.