Structural Insights and Functional Expansion of an Aerobic Skatole Synthase

SktA from the cyanobacterium Nostoc punctiforme NIES-2108 belongs to the versatile heme oxygenase-like domain-containing nonheme oxidase superfamily, whose natural function is to transform 5-bromotryptophan (5-Br-Trp) to 5-bromoskatole in an oxygen-dependent manner. As SktA is also capable of transforming tryptophan (Trp) to skatole, this enzyme represents a skatole synthase that operates under aerobic conditions. Here, the crystal structure of SktA and its complex with 5-Br-Trp and Trp are reported. The amino acids responsible for the substrate binding and the diiron cluster formation were revealed, and their roles in the catalytic reaction were examined via mutagenesis experiments. In addition, we found that SktA is capable of transforming 7-bromotryptophan and 5,7-dibromotryptophan to yield 7-bromoskatole and 5,7-dibromoskatole, respectively, and resolved the crystal structures of SktA in complex with these alternative substrates to elucidate the plasticity of the substrate-binding pocket. Finally, we show that combining SktA and regiospecific self-sufficient Trp halogenases in one-pot reaction systems efficiently transforms Trp to 5-bromo-7-iodo-skatole or 5-iodo-7-bromo-skatole. In summary, this study reveals the structural information, substrate scope, and potential applications in synthetic biology as a module to generate differentially halogenated skatoles of SktA.