| Azoxy bond[-N=N（-O）-]is an important chemical bond in liquid crystals,dyes,biological medicine and asymmetric synthetic intermediates.Especially,it plays an important role in the synthesis of high energy density materials,as it can significantly improve the materials’energy level by increasing their physical density and energy strength whilst concurrently decreasing their sensitivity for a better storage safety.However,the chemical synthesis of these type of compounds is challenged by various problems,such as the low synthetic efficiency,uncontrollable stereo and spatial selectivity,poor atom economy,and risky issue associated with synthetic safety.In overcoming these issues,the efficient biosynthesis of these promising compounds would clearly be of high benefit to the chemical industry.To date,a few azoxy natural products have been reported.Our laboratory have identified three aromatic azoxy natural products,azoxymycins A（5）,B（4）and C（3）,and their biosynthetic gene cluster from Streptomyces chattanoogensis.Interestingly,the deletion of azo C caused the accumulation of two amine precursors,but the biosynthetic mechanisms of azoxy bond from the amine precursors remains unclear.Reconstruction of azoxy bond biosynthesis with AzoC.In silico analysis indicated that AzoC belongs to the ferritin-like superfamily,and has 37%and 53%sequence identity to canonical nonheme diiron N-oxygenases Aur F and Cml I,respectively.The iron to enzyme ratio of AzoC was determined to be 1.78 using ferrozine assays.Native-PAGE and size exclusion chromatography analysis showed that purified recombinant AzoC had formed homodimer in solution.So it was propoesd that AzoC is a nonheme diiron N-oxygenase.To construct in vitro enzyme reaction of AzoC,we purified precursors 1 and 2 from culture broth of S.chattanoogensis（35）azo C mutant.HPLC analysis showed that 1 and 2 were both mixtures of spontaneously inter-convertible C4 E/Z isomers.So isomers consumption of 1 and 2 by AzoC was analyzed,and the biosynthetic pathway of azoxymycins was amended.Biosynthetic mechanism of azoxy bond.We tested other p-aminobenzene compounds and found that AzoC could convert p-aminobenzene acrylic acid（6）,p-amino benzoic acid,p-amino phenylacetic acid,p-amino phenylpropionic acid and p-amino phenylbutyric acid to their corresponding azoxy analogues.To explore the detailed biosynthetic pathway of azoxy bond,we chose 6 as a model substrate as it is structurally similar to 1 but has no E/Z isomerization.The hydroxylamine（7）,nitroso（8）and azoxy（9）analogs of 6 were chemically synthesized.Then both time-course and single turn-over in vitro experiments were performed.Time-course reactions seemed to suggest that 7 was the first oxidative intermediate in azoxy bond biosynthesis.However,single turn-over experiments indicated that 6 could only be transformed to 8 but not to 7.The results between time-course and single turn-over experiments were contradictory.So we compared two reaction systems,and found there was NADH in the time-course reaction system but not in single turn-over reaction system.It prompted us to assume that transformation between7 and 8 was a non-enzymatic reaction independent of AzoC.Then in vitro experiments conformed this hypothesis.The other redox pairs（NADP+/NADPH,FMN/FMNH2,FAD/FADH2,H2O2/sodium dithionite）could also mediate 8 and 7 transformation for subsequent azoxy bond formation.Biosynthetic scheme of 9 from 6 was proposed.AzoC enzymatically oxidizes 6 to 8,then NAD+/NADH induces non-enzymatic reciprocal transformation between 7 and 8.9 is then formed during the redox reaction process.Mechanism study of AzoC’s catalytic activity.The literature reported a new model of non-heme di-iron oxygenase based on theoretical calculation.It was proposed that the oxygenated intermediate centers（P）of Aur F and Cml I are ambiphilic hydroperoxo species.P acts as electrophilic reagent to oxidate amine to the nitroso group,and also acts as nucleophilic reagent to oxidate nitroso to the nitro group.The oxidation of amine to nitroso group in our study corresponds to the nucleophilic reactivity of Aur F and Cml I.We presumed that AzoC-P was not as nucleophilic as Aur F-P in proceeding the oxidization of the nitroso group to nitro.As the nucleophilic attack of P on the nitroso group was coupled with an electron-transfer to E196（glutamic acid 196）bonding Fe.The relevant Fe was bonded to the E198-H232 sequence of AzoC.So we constructed an AzoC mutant by replacing its E198-H232 sequence to the corresponding sequence of Aur F.AzoC-mutant was able to catalyze the oxidization of 8 to 10,indicating that the nucleophilic reactivity was partly restored.The modelling results implies that the E198’s binding model and relative location within the active center might be the mechanism behind AzoC’s nucleophilic deficiency in oxidizing the nitroso group to nitro.In conclusion,we have reported a unique azoxy bond biosynthetic mechanism composed of an enzymatic nitroso moiety producing reaction and a non-enzymatic N-N bond coupling reaction.The enzyme reaction mechanism was proposed using in silico modelling and docking experiments,Our mechanism discovery will be of high benefit to the biosynthesis of azoxy compounds.Especially,it will provide molecular basis for the biosynthesis of azoxy high energy density materials. |
