Tandem Action of the O2- and FADH2-Dependent Halogenases KtzQ and KtzR Produce 6,7-Dichlorotryptophan for Kutzneride Assembly

Abstract

Kutznerides are actinomycete-derived antifungal nonribosomal hexadepsipeptides which are assembled from five unsual nonproteinogenic amino acids and one hydroxy acid. Conserved in all structurally characterized kutznerides is a dichlorinated tricyclic hexahydropyrroloindole postulated to be derived from 6,7-dichlorotryptophan. In this Communication, we identify KtzQ and KtzR as tandem acting FADH₂-dependent halogenases that work sequentially on free l-tryptophan to generate 6,7-dichloro-l-tryptophan. Kinetic characterization of these two enzymes has shown that KtzQ (along with the flavin reductase KtzS) acts first to chlorinate at the 7-position of l-tryptophan. KtzR, with a ∼120 fold preference for 7-chloro-l-tryptophan over l-tryptophan, then installs the second chlorine at the 6-position of 7-chloro-l-tryptophan to generate 6,7-dichloro-l-tryptophan. These findings provide further insights into the enzymatic logic of carbon−chloride bond formation during the biosynthesis of halogenated secondary metabolites.