Molecular Dynamics Simulations Guide Chimeragenesis and Engineered Control of Chemoselectivity in Diketopiperazine Dimerases

In the biosynthesis of the tryptophan‐linked dimeric diketopiperazines (DKPs), cytochromes P450 selectively couple DKP monomers to generate a variety of intricate and isomeric frameworks. To determine the molecular basis for selectivity of these biocatalysts we obtained a high‐resolution crystal structure of selective Csp2−N bond forming dimerase, AspB. Overlay of the AspB structure onto C−C and C−N bond forming homolog NzeB revealed no significant structural variance to explain their divergent chemoselectivities. Molecular dynamics (MD) simulations identified a region of NzeB with increased conformational flexibility relative to AspB, and interchange of this region along with a single active site mutation led to a variant that catalyzes exclusive C−N bond formation. MD simulations also suggest that intermolecular C−C or C−N bond formation results from a change in mechanism, supported experimentally through use of a substrate mimic. The high‐resolution crystal structure of C−N bond forming diketopiperazine dimerase, AspB, was solved. However, the near complete superposition of active site residues and bound substrates in AspB/NzeB masked the molecular basis for their orthogonal chemoselectivities. Molecular dynamics simulations guided rational chimeragenesis to reprogram NzeB dimerase selectivity. Substrate mimics further validated differential substrate binding by the chemodivergent dimerases.