Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-L-methionine

Abstract

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 SAM-dependent 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.

Fig. 1.

Organization of the sal biosynthetic gene cluster from Salinispora tropica. The sal DNA sequence in strains CNB-476 and CNB-440 is 99% identical. Genes putatively involved in the chloroethylmalonyl-CoA pathway (red), construction of the core γ-lactam-β-lactone ring system (gray), assembly of the nonproteinogenic amino acid l-3-cyclohex-2′-enylalanine (blue), regulation and resistance (yellow), unknown (white), and 2 partial transposases (black) are color-coded.

Fig. 2.

Inactivation of the PKS gene salA completely abolishes salinosporamide (sal.) production. HPLC chromatograms of culture extracts with detection at 210 nm. Mt, mutant; wt, wild-type.

Fig. 3.

Comparison of chloroethylmalonyl-CoA and ethylmalonyl-CoA biosynthetic pathways. (A) Proposed pathway to chloroethylmalonyl-CoA as a PKS extender unit in salinosporamide A biosynthesis. (B) The corresponding ethylmalonyl-CoA moiety in salinosporamide B is derived from acetate (9). Crotonyl-CoA carboxylase/reductase (CCR) is a key enzyme in the ethylmalonyl-CoA pathway (19).

Fig. 4.

Chromatographic analysis of intermediates in the chloroethylmalonyl-CoA pathway. (A) 5-ClR as a key intermediate in the chloroethylmalonyl-CoA pathway. Scheme of the method used to derivatize 5-ClR for UV detection (352 nm) (Upper). LC/MS analysis of derivatized 5-ClR and water extracts of the salL (negative control) and salM mutants (Lower). (B) Feeding experiments with postulated intermediates (or their counterparts) in the chloroethylmalonyl-CoA pathway restore salinosporamide A production in the salL mutant. See Results for details.