p-aminobenzoate N-oxygenase:Novel Enzyme Chemistry for Antibiotics Biosynthesis
Many clinically important drugs are natural products produced by bacteria and fungi. In particular, polyketide antibiotics have been found to be an extremely rich source of biologically active compounds with a broad range of pharmaceutical activities. Aromatic nitro groups are relatively rare functional groups in natural products including polyketide antibiotics but are found in diverse types of important antibiotics, such as chloramphenicol, pyrrolnitrin, aureothin, azomycin, and rufomycin. Surprisingly, the biosynthesis of aromatic nitro groups is poorly understood. To date, only two enzymes have been shown to catalyze the formation of aromatic nitro groups, and the catalytic mechanism is virtually uncharacterized. p-nitrobenzoate N-oxygenase (AurF) is involved in the biosynthesis of polyketide antibiotic aureothin and catalyzes the formation of p-nitrobenzoic acid (pNBA) from p-aminobenzoic acid. However, AurF shares no sequence similarity with any other functionally characterized oxygenases. The native enzymatic activity has not been demonstrated in vitro, and its catalytic mechanism is unclear. The nature of the cofactor remains a controversy, although AurF is a metalloenzyme. In these aspects, understanding the molecular mechanism of AurF provides the opportunity to diversify the core structures of polyketides containing aromatic nitro groups and to develop tools for new drug discovery. Research professor Yoo Seong Choi, together with a research team at University of Illinois at Urbana- Champagne (Professor Huimin Zhao’s group), successfully characterized the molecular mechanism of AurF by their combined biochemical and structural analysis. First of all, they restored the native enzymatic activity in vitro using chemical and biological reductants, respectively. The crystal structure of AurF in the oxidized state and the cocrystal structure with its product pNBA were also determined. Then, they resolved the controversy on the nature of the cofactor in AurF. From these results, they concluded that AurF is a new class of non-heme di-iron monooxygenase that catalyzes unusual sequential oxidation of aminoarenes to nitroarenes via hydroxylamine and nitroso intermediates. They expect that the study will not only advance basic understanding of the formation of nitro groups in natural product biosynthesis, but also provide insights into the origin of nitro groups in many medically important natural products. This work can possibly suggest tools to generate polyketide antibiotics with improved biological and pharmacological properties and to synthesize important aromatic nitro compounds. In addition, the architecture of the di-iron center, unique chemistry of AurF and easy enzyme preparation make AurF an excellent model for further investigation of the molecular mechanism of di-oxygen activation of metalloenzymes and for the application of combinatorial biosynthesis and industrial biocatalysis. The research was published in May issue of Proceedings of the National Academy of Sciences, entitled, “In vitro reconstitution and crystal structure of p-aminobenzoate N-oxygenase (AurF) involved in aureothin biosynthesis”.
Professor Yoo Seong Choi
Department of Chemical Engineering