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 (T) 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 AdmI 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, AdmI, 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 AdmI. AdmF will also use either stereoisomer of phenylalanine or beta-Phe when presented on AdmA and AdmI, 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.
Magarvey
Nathan A
NA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Fortin
Pascal D
PD
Thomas
Paul M
PM
Kelleher
Neil L
NL
Walsh
Christopher T
CT
D000103:Acetyl-CoA Carboxylase / Q000037:antagonists & inhibitors; D000577:Amides / Q000378:metabolism; D049148:Aminoacylation; D004791:Enzyme Inhibitors / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism; D011090:Polyenes / Q000378:metabolism; D011758:Pyrroles / Q000378:metabolism; D013388:Succinimides / Q000378:metabolism; D011503:Transglutaminases / Q000378:metabolism
D000577:Amides; D004791:Enzyme Inhibitors; D011090:Polyenes; D011758:Pyrroles; D013388:Succinimides; C487819:moiramide B; C088069:andrimid; D010649:Phenylalanine; D011503:Transglutaminases; D000103:Acetyl-CoA Carboxylase