Polyketides are among the major classes of bioactive natural products used to treat microbial infections, cancer, and other diseases. Here we describe a pathway to chloroethylmalonyl-CoA as a polyketide synthase building block in the biosynthesis of salinosporamide A, a marine microbial metabolite whose chlorine atom is crucial for potent proteasome inhibition and anticancer activity. S-adenosyl-L-methionine (SAM) is converted to 5-chloro-5-deoxyadenosine (5-ClDA) in a reaction catalyzed by a SAMdependent chlorinase as previously reported. By using a combination of gene deletions, biochemical analyses, and chemical complementation experiments with putative intermediates, we now provide evidence that 5-ClDA is converted to chloroethylmalonyl-CoA in a 7-step route via the penultimate intermediate 4-chlorocrotonyl-CoA. Because halogenation often increases the bioactivity of drugs, the availability of a halogenated polyketide building block may be useful in molecular engineering approaches toward polyketide scaffolds.actinomycete ͉ biological halogenation ͉ marine natural product ͉ proteasome inhibitor ͉ Salinispora tropica
The salinosporamides comprise a natural product family of potent anticancer agents produced by the marine bacterium Salinispora tropica. This group of densely functionalized β-lactone-γ-lactam proteasome inhibitors is largely distinguished through structural differences at C-2 bearing methyl, ethyl, chloroethyl, and propyl substituents per salinosporamides D (1), B (2), A (3) and E (4), respectively (Scheme 1). [1][2][3] The recent discovery of the related metabolite cinnabaramide A (5) from a terrestrial streptomycete, 4 which instead harbors a C-2 hexyl chain, further extends the natural salinosporamide structural family. We recently reported that salinosporamides A and B are biosynthetic products derived from an unusual hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway initiated by the chain elongation of acetyl-S~ACP by chloroethylmalonyl-CoA or ethylmalonyl-CoA, 5,6 respectively, followed by the non-proteinogenic amino acid cyclohexenylalanine (Scheme 1). 7 The selection of the PKS extender unit is controlled by the acyltransferase domain AT 1 from the hexadomained SalA synthase. Herein we report that salinosporamides D and E are respectively accessed from methylmalonyl-CoA and propylmalonyl-CoA, the latter of which is a newly described PKS extender unit that belongs to a growing family of PKS substrates derived from α,β-unsaturated fatty acids.Based on the biosynthetic assembly of salinosporamide B from a butyrate building block via ethylmalonyl-CoA, 5,6 we reasoned that the methyl analog salinosporamide D (1) would be similarly assembled from a propionate unit via the common PKS substrate (2S)-methylmalonyl-CoA (Scheme 1). We explored this assumption by administering [1- 13 C] propionate to the S. tropica salL-deficient mutant, which is specifically unable to synthesize the chlorinated salinosporamide A as the major product of the sal pathway due to inactivated 5'-chloro-5'-deoxyadenosine synthase SalL. 8 Isolation and characterization of the resultant salinosporamide D by NMR revealed the specific 13 C-enrichment (5%) at C-1, thereby confirming its assembly from propionate.We next turned our attention to the propyl analog salinosporamide E (4), which would presumably derive from a pentanoate building block. If so, this would imply the prospect of a new PKS building block, namely propylmalonyl-CoA. Salinosporamide E was similarly isolated from the [1-13 C]propionate feeding experiment, and NMR analysis confirmed 13 Cenrichment (40%) at C-12, thereby suggesting an origin from pentanoate derived from bsmoore@ucsd.edu.
The DEBS1-TE fusion protein is comprised of the loading module, the first two extension modules, and the terminal TE domain of the Saccharopolyspora erythraea 6-deoxyerythronolide B synthase. DEBS1-TE produces triketide lactones that differ on the basis of the starter unit selected by the loading module. Typical fermentations with plasmid-based expression of DEBS1-TE produce a 6:1 ratio of propionate to isobutyrate-derived triketide lactones. Functional dissection of the loading module from the remainder of DEBS1-TE results in 50% lower titers of triketide lactone and a dramatic shift in the production to a 1:4 ratio of propionate to isobutyrate-derived products. A series of radiolabeling studies of the loading module has shown that transfer from the AT to the ACP occurs much faster for propionate than for isobutyrate. However, the equilibrium occupancy of the AT favors isobutyrate such that propionate is outcompeted for ACP occupancy. Thus, propionyl-ACP is the kinetic product, while isobutyryl-ACP is the thermodynamic product. A slowed transfer from the loading domain ACP to first-extension module KS due to functional dissection of DEBS1-TE allows this isobutyryl-ACP-favored equilibrium to be realized and likely accounts for the observed shift in triketide lactone products.
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