Characterization of the Oxazinomycin Biosynthetic Pathway Revealing the Key Role of a Nonheme Iron-Dependent Mono-oxygenase.
Daan RenYu-Hsuan LeeShao-An WangHung-Wen LiuPublished in: Journal of the American Chemical Society (2022)
Oxazinomycin is a C -nucleoside natural product with antibacterial and antitumor activities. In addition to the characteristic C -glycosidic linkage shared with other C -nucleosides, oxazinomycin also features a structurally unusual 1,3-oxazine moiety, the biosynthesis of which had previously been unknown. Herein, complete in vitro reconstitution of the oxazinomycin biosynthetic pathway is described. Construction of the C -glycosidic bond between ribose 5-phosphate and an oxygen-labile pyridine heterocycle is catalyzed by the C -glycosidase OzmB and involves formation of an enzyme-substrate Schiff base intermediate. The DUF4243 family protein OzmD is shown to catalyze oxygen insertion and rearrangement of the pyridine C -nucleoside intermediate to generate the 1,3-oxazine moiety along with the elimination of cyanide. Spectroscopic analysis and mutagenesis studies indicate that OzmD is a novel nonheme iron-dependent enzyme in which the catalytic iron center is likely coordinated by four histidine residues. These results provide the first example of 1,3-oxazine biosynthesis catalyzed by an unprecedented iron-dependent mono-oxygenase.