Many heterocyclic aromatic N-oxides have potent biological activities, but the mechanism for the biosynthesis of these compounds is often unclear. We recently isolated six antibacterial compounds from Lysobacter antibioticus OH13 and determined their structures to be phenazines, including the aromatic N-oxide myxin, a well-known antibiotic. With the goal of understanding the multiple N-oxidations and O-methylations found in these compounds, we sequenced the OH13 genome and identified a 10-gene cluster (LaPhz) and showed that LaPhz genes were required for phenazine biosynthesis. LaPhzNO1 in the cluster was homologous to genes encoding cyclohexanone monooxygenases, flavoproteins which catalyze a Baeyer-Villiger type oxidation. Mutation of LaPhzNO1 abolished production of all N-oxides, while production of two non-oxide phenazines markedly increased. We expressed LaPhzNO1 in E. coli and demonstrated that the enzyme is an NADPH-dependent, flavin N-monooxygenase. We further showed that LaPhzNO1 together with LaPhzS, another flavin monooxygenase, converted phenazine 1,6- dicarboxylic acid (PDC) to 1,6-dihydroxyphenazine N5,N10-dioxide. LaPhzNO1 thus represents the first experimentally characterized N-monooxygenase in the biosynthesis of heterocyclic aromatic natural products. In addition, we expressed LaPhzM in E. coli and showed that the purified enzyme LaPhzM was able to catalyze phenazine O-methylation and N-methylation using S-adenosylmethionine as co-substrate. These results establish the enzymatic mechanisms for phenazine core decorations, which could lead to hundreds of phenazine natural products.

Enzymatic mechanisms for decorating phenazine antibiotics from Lysobacter antibioticus

253rd National Meeting (2017)

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