Chemical ecology: Reprogramming a termite monarchy.

authors identified a tyrosinase-like enzyme, NspF, that was homologous to tyrosinaselike GriF in grixazone biosynthesis. Tyrosinases contain a binuclear copper cluster and have known functions as monoand diphenol oxidases (for example, in melanin production), in keeping with the elucidated role of GriF. However, because a traditional N-oxidase was absent from the 4,3-HNBAm gene cluster, the authors further hypothesized that NspF was a nitrosation copper enzyme similar to tyrosinases. Indeed, NspF catalyzed nitrosation of its proposed biosynthetic substrate 3-amino-4-hydroxybenzamide to 4,3-HNBAm in vitro, and NspF single and double mutants in which one or two conserved histidines essential to copper binding were replaced by asparagines showed decreased and abolished nitrosoforming activity, respectively. Additionally, a copper chaperone, NspE, which might provide copper to the NspF active site, was required for in vivo activity. An interesting question regarding the presented nitrosation pathway is how overoxidation of the nitrogen is prevented by NspF. A plausible mechanism for the controlled nitrosation of 3-amino-4hydroxybenzamide (3,4-AHBAm) that would prevent overoxidation is shown in Figure 1b. In this mechanism, the active site contains a peroxo–dicopper(ii) complex that is coordinated by the phenol substrate to one copper atom during the oxidation mechanism8,9. The nitrosation mechanism would involve a radical recombination of the amine with both oxygens in the peroxo–dicopper(ii) complex, leading to intermediate B (Fig. 1b), and subsequent elimination would result in water and the formation of stable 4,3-HNBAm; the necessity for the reactive oxygen species to be regenerated would then facilitate release of the product at the nitroso oxygenation stage. Finally, the enzyme is positioned to pick up another oxygen molecule for the next catalytic cycle. Further comparison of NspF and GriF, as well as NspF and the previously identified RubN8, should yield additional insights into this mechanism as well as the discrimination afforded by these enzymes. This study presents an exciting addition to the existing N-oxidation mechanisms, as both the first reconstitution of a secondary metabolic pathway to a nitroso natural product and the concomitant discovery of a new copper-dependent N-oxidase, NspF. Ultimately, the characterization of NspF fills in a gap in our understanding of how biology decorates aromatic rings with nitric oxide. Unlike any other pathways described to date, NspF carries out controlled aromatic C-nitrosation biochemistry, introduces new copper-dependent biocatalysis and, in so doing, implies that there is broader versatility among copper enzymes in the biosynthesis of biologically active natural products than had previously been demonstrated. Aromatic C-nitrososynthases, much like tyrosinases, may soon be used extensively in synthetic reactions that carry out aromatic C-nitrosations. ■