Rational Mining and Engineering of Unique P450 Enzymes Enable Production of Diverse Dimeric Tryptophan-Containing Diketopiperazine Alkaloids

Dimeric tryptophan-containing diketopiperazine (DTDKP) alkaloids exhibit unique structures and promising bioactivities. Bacterial P450 enzymes can effectively biosynthesize diverse asymmetric DTDKPs, but their narrow substrate scope, primarily limited to small tryptophan-containing diketopiperazines (TDKPs), severely restricts structural diversity. To address this limitation, three P450 TDKP dimerases (StsuP450, AcolP450, and StnoP450) that accept bulky substrates such as cyclo-l-Trp-l-Trp (cWW) were identified through genome mining guided by near-attack conformation (NAC) analysis. Among them, StsuP450, exhibiting the highest selectivity, was further subjected to protein engineering. Computer analysis identified A398 and W184 as key residues governing substrate selectivity in the upper binding pocket. Subsequent rational mutagenesis at these positions broadened the catalytic versatility toward both bulky and small TDKPs. Molecular dynamics simulation revealed the molecular basis of variants A398F and W184F/L in regulating the substrate binding via directly altering pocket volume and inducing loop rearrangement, respectively. Utilizing the three unique P450s and the StsuP450 mutants, we characterized 13 new homodimeric DTDKPs with diverse C3/N1–aryl couplings and generated at least 46 heterodimers (five structurally characterized). The rational mining and engineering approaches provide important guidelines in future enzyme discovery and biosynthesis of valuable natural products.