An engineered N-acyltransferase-LOV2 domain fusion protein enables light-inducible allosteric control of enzymatic activity

Abstract

Transferases are ubiquitous across all known life. While much work has been done to understand and describe these essential enzymes, there have been minimal efforts to exert tight and reversible control over their activity for various biotechnological applications. Here, we apply a rational, computation-guided methodology to design and test a transferase-class enzyme allosterically regulated by light-oxygen-voltage 2 sensing domain. We utilize computational techniques to determine the intrinsic allosteric networks within N-acyltransferase (Orf11/∗Dbv8) and identify potential allosteric sites on the protein's surface. We insert light-oxygen-voltage 2 sensing domain at the predicted allosteric site, exerting reversible control over enzymatic activity. We demonstrate blue-light regulation of N-acyltransferase (Orf11/∗Dbv8) function. Our study for the first time demonstrates optogenetic regulation of a transferase-class enzyme as a proof-of-concept for controllable transferase design. This successful design opens the door for many future applications in metabolic engineering and cellular programming.

Keywords: DMD; allostery; discrete molecular dynamics; enzymology; optogenetics; protein engineering.