標題: Biochemical characterization and substrate specificity of the gene cluster for biosyntheses of K-252a and its analogs by in vitro heterologous expression system of Escherichia coli
作者: Chiu, Hsien-Tai
Lin, Yu-Chin
Lee, Meng-Na
Chen, Yi-Lin
Wang, Mei-Sin
Lai, Chia-Chun
生物科技學系
Department of Biological Science and Technology
公開日期: 2009
摘要: The indolocarbazole family of natural products has attracted great attention because of their unique structural features and potential therapeutic applications. Structurally distinct in the family, K-252a is characterized by an unusual dihydrostreptose moiety cross-bridged to K-252c aglycone with two C-N linkages. K-252a has served as a valuble lead for treatments of various cancers and neurodegenerative disorders. Recent cloning of the nok gene cluster for biosyntheses of K-252a and its analogs from Nocardiopsis sp. K-252 (NRRL15532) has revealed the nokABCD genes indispensible for K-252c biosynthesis and the key gene (nokL) coding for N-glycosylation. Herein, we report the first, successful demonstration of in vitro sugar transferase activity of indolocarbazole N-glycosyltransferase (NokL) by use of soluble protein expressed from Escherichia coli. Notably, NokL was found to exhibit peculiar mode of substrate promiscuity. Moreover, NokA and NokB reactions were biochemically characterized thoroughly by natual and alternative (e. g. fluoro-) substrates and by ammonium hydroxide (NH(4)OH). Interestingly, the in vitro expression of NokA revealed high substrate stereoselectivity, giving several indole-3-pyruvic acid-derived compounds, including indol-3-carboxaldehyde (ICA) and indole-3-acetic acid. The use of NH(4)OH successfully dissected the in vitro NokA/NokB coupled reaction, revealing mechanistic insight into the enzymes and their cross-talking relationship. Also, a simple, useful method to synthesize K-252d, ICA and chromopyrrolic acid (the NokB product) was developed by the E. coli expression systems of NokL, NokA and NokA/NokB, respectively. Together with NokA and NokB, NokL may serve as a useful tool for combinatorial engineering of K-252a and its analogs for improved therapeutic values.
URI: http://hdl.handle.net/11536/7800
http://dx.doi.org/10.1039/b912395b
ISSN: 1742-206X
DOI: 10.1039/b912395b
期刊: MOLECULAR BIOSYSTEMS
Volume: 5
Issue: 10
起始頁: 1192
結束頁: 1203
顯示於類別:期刊論文


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