Gatekeeping versus promiscuity in the early stages of the andrimid biosynthetic assembly line

The antibiotic andrimid, a nanomolar inhibitor of bacterial acetyl coenzyme A carboxylase, is generated on an unusual polyketide/nonribosomal peptide enzyme assembly line in that all thiolation M domains/small-molecule building stations are on separate proteins. In addition, a transglutaminase homologue is used to condense andrimid building blocks together on the andrimid assembly line. The first two modules of the andrimid assembly line yields an octatrienoyl-beta-Phe-thioester tethered to the Adml T domain, with amide bond formation carried out by a free-standing transglutaminase homologue AdmF. Analysis of the aminomutase AdmH reveals its specific conversion from L-Phe to (s)-beta-Phe, which in turn is activated by Admj and ATP to form (S)-beta-Phe-aminoacyl-AMP. Admj then transfers the (S)-beta-Phe moiety to one of the free-standing T domains, Adml, but not AdmA, which instead gets loaded with an octatrienoyl group by other enzymes. AdmF, the amide synthase, will accept a variety of acyl groups in place of the octatrienoyl donor if presented on either AdmA or Adml. AdmFwill also use either stereoisomer of phenylatanine or beta-Phe when presented on AdmA and Adml, but not when placed on noncognate T domains. Further, we show the polyketide synthase proteins responsible for the polyunsaturated acyl cap can be bypassed in vitro with N-acetylcysteamine as a low-molecular-weight acyl donor to AdmF and also in vivo in an Escherichia coli strain bearing the andrimid biosynthetic gene cluster with a knockout in admA.