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.

Identification of a Halogenase Involved in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius

“…Genes for flavin-dependent halogenase have been found in a number of prokaryotic and fungal biosynthetic gene clusters for secondary metabolites (21). Halogenated natural products are widely distributed in nature, and many of them show biological activity as antibacterials, antifungals, and anticancer agents (39)(40)(41). Investigation of biosynthesis of halogenated compounds is interesting also for the biotechnological potential of these enzymes, in particular of FADH 2 -dependent halogenases.…”

pH-dependent Studies Reveal an Efficient Hydroxylation Mechanism of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

“…Reactions catalyzed by two-component monooxygenases include oxygenation and halogenation of organic compounds such as p-hydroxyphenylacetate (9), phenol (10), trichorophenol (11,12), p-nitrophenol (13), styrene (14 -16), alkane sulfonate (17,18), and EDTA (19). Two-component monooxygenases are also involved in oxygenation and halogenation reactions in the biosynthetic pathways of actinorhodin (ActVA) (20), angucyclin (21), enediyne (SgcC) (22), rebeccamycin (RebH) (23), pyrrolnitrin (PrnA) (24), violacein (25), kutzneride (26), and differentiation-inducing factor-1 (27).…”

An Unusual Flavin-Dependent Halogenase from the Metagenome of the Marine Sponge Theonella swinhoei WA

“…To our knowledge, the only other FDTH with a preference for a substituted tryptophan is KtzR, which installs chlorine at the 6-position of 7-chlorotryptophan in the biosynthesis of kutzneride. 36 Encouraged by these initial results, we extended our study of the substrate scope of KrmI. Again we utilized crude lysate containing the full-length protein in incubations with a variety of potential substrates (Figure 8).…”