Acetyl-CoA-Mediated Post-Biosynthetic Modification of Desferrioxamine B Generates N - and N- O -Acetylated Isomers Controlled by a pH Switch.

Biosynthesis of the hydroxamic acid siderophore desferrioxamine D 1 (DFOD 1 , 6 ), which is the N -acetylated analogue of desferrioxamine B (DFOB, 5 ), has been delineated. Enzyme-independent Ac-CoA-mediated N -acetylation of 5 produced 6 , in addition to three constitutional isomers containing an N- O -acetyl group installed at either one of the three hydroxamic acid groups of 5 . The formation of N -Ac-DFOB (DFOD 1 , 6 ) and the composite of N- O -acetylated isomers N- O -Ac-DFOB[001] ( 6a ), N- O -Ac-DFOB[010] ( 6b ), and N- O -Ac-DFOB[100] ( 6c ) (defined as the N- O -Ac motif positioned within the terminal amine, internal, or N -acetylated region of 5 , respectively), was pH-dependent, with 6a - 6c dominant at pH < 8.5 and 6 dominant at pH > 8.5. The trend in the pH dependence was consistent with the p K a values of the NH 3 + (p K a ∼ 10) and N-OH (p K a ∼ 8.5-9) groups in 5 . The N - and N- O -acetyl motifs can be conceived as a post-biosynthetic modification (PBM) of a nonproteinaceous secondary metabolite, akin to a post-translational modification (PTM) of a protein. The pH-labile N- O -acetyl group could act as a reversible switch to modulate the properties and functions of secondary metabolites, including hydroxamic acid siderophores. An alternative (most likely minor) biosynthetic pathway for 6 showed that the nonribosomal peptide synthetase-independent siderophore synthetase DesD was competent in condensing N' -acetyl- N -succinyl- N -hydroxy-1,5-diaminopentane ( N' -Ac-SHDP, 7 ) with the dimeric hydroxamic acid precursor (AHDP-SHDP, 4 ) native to 5 biosynthesis to generate 6 . The strategy of diversifying protein structure and function using PTMs could be paralleled in secondary metabolites with the use of PBMs.