Characterization of the Oxazinomycin Biosynthetic Pathway Revealing the Key Role of a Nonheme Iron-Dependent Mono-oxygenase

Abstract

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.

Unusual Dioxygen-Dependent Reactions Catalyzed by Nonheme Iron Enzymes in Natural Product Biosynthesis

“…194,195 The reaction of OzmD produces cyanide as a byproduct in addition to 163, as confirmed by a chemical derivatization method. 195 The released cyanide molecule originates from C3− NH 2 of 162, as verified by in vitro assays with 13 C-or 15 N-labeled substrates, which is in good agreement with an early feeding experiment with [2-14 C]-L-Glu. Furthermore, a series of 18 O labeling experiments indicated that the two carbonyl oxygens in 163 are retained during the OzmD-catalyzed reaction, while the alkoxy oxygen of 163 is derived from O 2 .…”

“…The unique pyridine base is derived from l -glutamine via the sugar-linked aminopyridine 162 as the intermediate. In vitro assays demonstrated that the unusual rearrangement reaction of 162 to 163 is catalyzed by OzmD. , …”

“…Based on these observations, a plausible mechanism for the rearrangement reaction was proposed (Scheme B) . Although the oxidation state of iron during the reaction is unclear, it may bind to 162 and O 2 to initiate the catalysis.…”

“…The sequence analysis revealed that OzmD exhibits similarity to GedK, as both enzymes are members of the DUF4243 family. 195 A structure prediction and mutagenesis study suggested that the four histidine residues in the active site, namely H30, H127, H268, and H352, are critical for the activity of OzmD, implying their roles in binding the iron for catalysis.…”

“…Based on these observations, a plausible mechanism for the rearrangement reaction was proposed (Scheme 19B). 195 Although the oxidation state of iron during the reaction is unclear, it may bind to 162 and O 2 to initiate the catalysis. The iron-mediated electron transfer from 164 to O 2 generates the semiquinone radical−Fe−superoxo species 165.…”