Fig. 4: Proposed biosynthetic pathway of cispentacin.

All reaction steps of the biosynthetic pathway from 2-OG to cispentacin were biochemically elucidated. Initially, 2-OG is modified to 2-oxoglutaryl-AmcB (1-AmcB) by AmcH adenylate-forming acyltransferase. 1-AmcB then condenses with the elongation substrate malonyl-AmcB, followed by C2–C6 carbon bond formation and dehydration, which presumably involves residues on the enzyme acting as an acid and a base to form 2-AmcB. This series of reactions is catalyzed by the AmcF–AmcG heterodimer. 2-AmcB is subsequently decarboxylated by the action of AmcE and converted to 3-AmcB. Then, the FAS enzyme FabI, which is presumably recruited to AmcB, catalyzes the reduction of 3-AmcB to form 4-AmcB in the presence of NADH. Finally, 4-AmcB is aminated to 5-AmcB by AmcC aminotransferase in the presence of L-ornithine or L-lysine and then released from AmcB by AmcD-catalyzed hydrolysis to yield cispentacin.