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

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 Fe^II-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 Fe^II-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.