Abstract
The azoxy compounds with an intriguing chemical bond [-N=N+(-O−)-] are known to have broad applications in many industries. Our previous work revealed that a nonheme diiron N-oxygenase AzoC catalyzed the oxidization of amino-group to its nitroso analogue in the formation of azoxy bond in azoxymycins biosynthesis. However, except for the reported pyridine alkaloid azoxy compounds, most azoxy bonds of nitrogen heterocycles have not been biosynthesized so far, and the substrate scope of AzoC is limited to p-aminobenzene-type compounds. Therefore, it is very meaningful to use AzoC to realize the biosynthesis of azoxy nitrogen heterocycles compounds. In this work, we further studied the catalytic potential of AzoC toward nitrogen heterocycle substrates including 5-aminopyrimidine and 5-aminopyridine compounds to form new azoxy compounds through directed evolution. We constructed a double mutant L101I/Q104R via molecular engineering with improved catalytic efficiency toward 2-methoxypyrimidin-5-amine. These mutations also proved to be beneficial for N-oxygenation of methyl 5-aminopyrimidine-2-carboxylate. The structural analysis showed that relatively shorter distance between the substrate and the diiron center and amino acid residues of the active center may be responsible for the improvement of catalytic efficiency in L101I/Q104R. Our results provide a molecular basis for broadening the AzoC catalytic activity and its application in the biosynthesis of azoxy six-membered nitrogen catenation compounds.
Highlights
Published: 27 January 2022Natural azoxy compounds share a common functional group with the general structure [-N=N+ (-O− )-], which have been found in bacteria, fungi, plants, and marine sponge [1,2].According to the metabolism pathway, natural azoxy compounds are mainly involved in three classes including polyketides, shikimate-derived and amino acid-derived compounds.The special azoxy bond endows these compounds with multiple functionalities, especially with cytotoxic, nematocidal, and antimicrobial activity [3]
As the core structure of 5-aminopyrimidine compounds is similar to six-membered nitrogen catenation, this study provides a molecular basis for the biosynthesis of azoxy six-membered nitrogen catenation compounds
When the benzene ring was changed with the pyrimidine and pyridine rings, AzoC showed different catalytic activity according to different amino group positions
Summary
Published: 27 January 2022Natural azoxy compounds share a common functional group with the general structure [-N=N+ (-O− )-], which have been found in bacteria, fungi, plants, and marine sponge [1,2].According to the metabolism pathway, natural azoxy compounds are mainly involved in three classes including polyketides, shikimate-derived and amino acid-derived compounds.The special azoxy bond endows these compounds with multiple functionalities, especially with cytotoxic, nematocidal, and antimicrobial activity [3]. The azoxy bond is proven to be an effective structural unit to improve the detonation performance of nitrogen catenation compounds [8]. At present, azoxy compounds, especially azoxy nitrogen catenation compounds, are mainly chemically synthesized, which is performed under harsh reaction conditions, such as high pressure, application of a metal catalyst and high temperature [6,9]. The biosynthesis of these azoxy compounds may lead to an option for reactions performed under mild conditions. Pyridine alkaloid azoxy compounds from the natural origin have been reported [10,11,12], the biosynthesis of most
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