Abstract
Antimycins are anticancer compounds produced by a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. The biosynthesis of these compounds is well characterized, with the exception of the standalone β-ketoreductase enzyme AntM that is proposed to catalyze the reduction of the C8 carbonyl of the antimycin scaffold. Inactivation of antM and structural characterization suggested that rather than functioning as a post-PKS tailoring enzyme, AntM acts upon the terminal biosynthetic intermediate while it is tethered to the PKS acyl carrier protein. Mutational analysis identified two amino acid residues (Tyr185 and Phe223) that are proposed to serve as checkpoints controlling substrate access to the AntM active site. Aromatic checkpoint residues are conserved in uncharacterized standalone β-ketoreductases, indicating that they may also act concomitantly with synthesis of the scaffold. These data provide novel mechanistic insights into the functionality of standalone β-ketoreductases and will enable their reprogramming for combinatorial biosynthesis.
Highlights
Antimycins are anticancer compounds produced by a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway
The hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) biosynthetic gene cluster (BGC) responsible for the production of antimycins has been reported,[5] and the proposed biosynthetic pathway is shown in Figure 1.6−8 A key step in the biosynthesis of antimycin is the reduction of the C8 carbonyl by a standalone β-ketoreductase (KR) named AntM, which is required for the subsequent diversification of the position by AntB, an acyltransferase.[9]
Liquid chromatography−high-resolution mass spectrometry (LC-HRMS) analysis of chemical extracts resulting from this ΔantM strain revealed that it no longer produced 1−4, and antimycins harboring a C8 carbonyl or their corresponding unreduced linear intermediates were not detected (Figure S1)
Summary
Antimycins are anticancer compounds produced by a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. These regions were recently shown to be important for another tetracyclic aromatic C6-ketoreductase, LugOII (lugdunomycin).[14] Sequence alignment of AntM with UrdMred and LanV proteins shows that the R1−R4 regions around the active site are the regions that diverge most between UrdMred/LanV and AntM (Figure 2C,D), suggesting that these portions of the protein are likely to determine the substrate specificity of AntM.
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