Microbially-sourced secondary metabolites that contain a central 2,5-diketopiperazine (DKP) scaffold represent an expanding class of natural products that show promise as therapeutic targets in the treatment of human disease. Often, biological molecules that contain a DKP core are derived from cyclic dipeptides (CDPs). Cytochrome P450s (CYPs) are prevalently found in CDP pathways and have been shown to catalyze the homodimerization of two identical DKP-dipeptides, or in some cases, the heterodimerization of a single DKP-dipeptide with a free nucleotide. Our previous studies have shown that the provision of a single electron and O₂ can effectively support different modes of DKP homodimerization. To assess whether this atypical CYP reaction trajectory extends to nucleobase transfer by heterodimerases, we have characterized a CYP (termed GutD) that orchestrates the transfer of guanine to the indole moiety of cyclo-L-Trp-L-Trp (cWW) to produce guanitrypmycin C3-1. Structural, spectroscopic, and calorimetric studies reveal the binding order and specificity of substrates to GutD and rationalizes the prevalence of guanylated GutD products isolated in vivo. The analysis of co-substrate requirements reveals branched pathways for heterodimerization with alternative substrates. Finally, the mechanism and substrate scope of GutD is leveraged to produce various guanitrypmycin derivatives in vitro and in cellulo.