Biosynthesis of streptolidine involved two unexpected intermediates produced by a dihydroxylase and a cyclase through unusual mechanisms

Chin-Yuan Chang, Syue Yi Lyu, Yu Chen Liu, Ning Shian Hsu, Chih Chung Wu, Cheng Fong Tang, Kuan Hung Lin, Jin Yuan Ho, Chang Jer Wu, Ming Daw Tsai, Tsung Lin Li*

*Corresponding author for this work

Research output: Contribution to journalArticle

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Abstract

Streptothricin-F (STT-F), one of the early-discovered antibiotics, consists of three components, a β-lysine homopolymer, an aminosugar D-gulosamine, and an unusual bicyclic streptolidine. The biosynthesis of streptolidine is a long-lasting but unresolved puzzle. Herein, a combination of genetic/biochemical/structural approaches was used to unravel this problem. The STT gene cluster was first sequenced from a Streptomyces variant BCRC 12163, wherein two gene products OrfP and OrfR were characterized in vitro to be a dihydroxylase and a cyclase, respectively. Thirteen high-resolution crystal structures for both enzymes in different reaction intermediate states were snapshotted to help elucidate their catalytic mechanisms. OrfP catalyzes an FeII-dependent double hydroxylation reaction converting L-Arg into (3R,4R)-(OH)2-L-Arg via (3S)-OH-L-Arg, while OrfR catalyzes an unusual PLP-dependent elimination/addition reaction cyclizing (3R,4R)-(OH) 2-L-Arg to the six-membered (4R)-OH-capreomycidine. The biosynthetic mystery finally comes to light as the latter product was incorporation into STT-F by a feeding experiment. Green label: In streptolidine biosynthesis, OrfP catalyzes the FeII-dependent double hydroxylation of L-Arg to form 1, which is undergoes a PLP-dependent cyclization reaction catalyzed by OrfR to form 2. The longstanding question of the streptolidine biosynthesis was solved by the incorporation of [13C6]-2 (green dots are 13C) into streptothricin by a feeding experiment.

Original languageEnglish
Pages (from-to)1943-1948
Number of pages6
JournalAngewandte Chemie - International Edition
Volume53
Issue number7
DOIs
StatePublished - 10 Feb 2014

Keywords

  • biosynthesis
  • enzymes
  • isotopic labeling
  • protein structures
  • reaction mechanisms

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    Chang, C-Y., Lyu, S. Y., Liu, Y. C., Hsu, N. S., Wu, C. C., Tang, C. F., Lin, K. H., Ho, J. Y., Wu, C. J., Tsai, M. D., & Li, T. L. (2014). Biosynthesis of streptolidine involved two unexpected intermediates produced by a dihydroxylase and a cyclase through unusual mechanisms. Angewandte Chemie - International Edition, 53(7), 1943-1948. https://doi.org/10.1002/anie.201307989