In B. anthracis, petrobactin is produced by a nonribosomal peptide synthetase independent siderophore (NIS) synthetase pathway. The enzyme sequences used are anthrax siderophore biosynthesis (Asb) A through F, in alphabetical order. These gene clusters are identical to those used in M. hydrocarbonclasticus biosynthesis of petrobactin. In A. macleodii only the first three gene clusters, AsbA through AsbC, are identical to B. anthracis; then a longer AsbD and AsbF is next, followed by two hypothetical protein domains and a PepSY domain-containing gene. A. macleodii ends its sequence with AsbE.
The biosynthesis of petrobactin in B. anthracis can progress in order AsbA-AsbB-AsbE-AsbE or AsbA-AsbE-AsbB-AsbE.
If the enzymation reactions in this pathway proceed generally, in domains AsbA and AsbB the phosphorylation of a carboxylic acid forms an acylphosphate intermediate, which is then dephosphorylated by a primary amine in spermidine. In domain AsbE the lone pair of electrons on a primary amine allows for a nucleophilic attack on the electrophilic hydroxyl carbon. The sulfur on AsbE is protonated to form a thiol and the amide nitrogen is deprotonated.
The dehydration of 3-dehydroshikimic acid might proceed as a modified, enzyme-catalyzed dienol benzene rearrangement and reduction, leading to aromatization of the ring.