Characterization of the flavoenzyme XiaK as an N-hydroxylase and implications in indolosesquiterpene diversification
Qingbo Zhang1, Huixian Li1,2, Lu Yu3,4
1CAS Key Laboratory of Tropical Marine Bio-resources and Ecology , Guangdong Key Laboratory of Marine Materia Medica , South China Sea Institute of Oceanology , Chinese Academy of Sciences , 164 West Xingang Road , Guangzhou 510301 , China . Email: czhang2006@gmail.com ;
Abstract:
Flavoenzymes are ubiquitous in biological systems and catalyze a diverse range of chemical transformations. The flavoenzyme XiaK from the biosynthetic pathway of the indolosesquiterpene xiamycin A is demonstrated to mediate the in vivo biotransformation of xiamycin A into multiple products, including a chlorinated adduct as well as dimers characterized by C-N and N-N linkages that are hypothesized to form via radical-based mechanisms. Isolation and characterization of XiaK in vitro shows that it acts as a flavin-dependent N-hydroxylase that catalyzes the hydroxylation of xiamycin A at the carbazole nitrogen to form N-hydroxyxiamycin, a product which was overlooked in earlier in vivo experiments because its chemical and chromatographic properties are similar to those of oxiamycin. N-Hydroxyxiamycin is shown to be unstable under aerobic conditions, and characterization by electron paramagnetic resonance spectroscopy demonstrates formation of an N-hydroxycarbazole radical adduct. This radical species is proposed to serve as a key intermediate leading to the formation of the multiple xiamycin A adducts. This study suggests that non-enzyme catalyzed reactions may play a greater role in the biosynthesis of natural products than has been previously recognized.
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