Introduction: Enzymatic functionalization of aliphatic carbons is a potent strategy in sustainable organic synthesis. Our focus is on discovering and characterizing new alpha-ketoglutarate-dependent halogenases/hydroxylases (AKGHs) capable of functionalizing sp³ carbons in N-heterocycles, with wide-ranging applications in pharmaceuticals, agrochemicals, and fine chemicals.

Goals: Our objectives include discovering novel catalytic pathways by identifying and characterizing AKGHs for unexplored sp³ carbon functionalization, thereby revealing new sustainable routes for chemical transformations. We aim to elucidate the complexities of enzyme-substrate interactions, reaction kinetics and selectivity within N-heterocyclic compounds. We also plan to broaden the catalytic horizons of AKGHs, expanding their substrate scope via protein engineering to enhance enzyme versatility for a variety of chemical substrates.

Methods: Our approach leveraged computational tools, such as Hidden Markov Models and AlphaFold2, for AKGH identification and structural insights. We used analytical techniques such as GC, HPLC, MS, and NMR to monitor the kinetics, identify products, and determine stereochemistry. Additionally, we implemented protein engineering methods like site saturation mutagenesis (SSM) and random mutagenesis to improve enzyme properties and expand substrate scope.

Results: Our computational screening successfully identified ten novel AKGH candidates. Through optimization of heterologous production and reaction conditions, we have achieved efficient production of these enzymes. Initial tests reveal promising catalytic potential, with these AKGHs demonstrating the ability to facilitate selective hydroxylations and halogenations in specific N-heterocyclic substrates. Current protein engineering efforts are focused on broadening their substrate scope, enhancing their applicability in diverse chemical reactions.

Summary: Our research advances AKGH understanding, revealing new catalytic pathways and enhancing substrate versatility. These enzymes offer an eco-friendly approach to chemical synthesis, aligning with green chemistry principles for a more sustainable future.