Biosynthesis of Streptolidine Involved Two Unexpected Intermediates Produced by a Dihydroxylase and a Cyclase through Unusual Mechanisms

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 Fe II ‐dependent double hydroxylation reaction converting L ‐Arg into (3 R ,4 R )‐(OH) 2 ‐ L ‐Arg via (3 S )‐OH‐ L ‐Arg, while OrfR catalyzes an unusual PLP‐dependent elimination/addition reaction cyclizing (3 R ,4 R )‐(OH) 2 ‐ L ‐Arg to the six‐membered (4 R )‐OH‐capreomycidine. The biosynthetic mystery finally comes to light as the latter product was incorporation into STT‐F by a feeding experiment.