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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.

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.