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 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

Mining the Cinnabaramide Biosynthetic Pathway to Generate Novel Proteasome Inhibitors

“…Because the overall structure of cinnabaramides is suggestive of a biosynthetic origin based upon PKS and NRPS machinery and because the gene cluster for salinosporamides (sal) is known, [18] a cosmid library was constructed from the genome of strain JS360 and screened for the corresponding gene cluster in an approach based on hybridization with degenerated PKS and NRPS (as described in the Experimental Section).…”

“…Proposed function Sequence identity/similarity to [%] [a] Accession number Sequence identity/similarity to Salinispora tropica CNB-440 [18] [%] [b] cinA extender units of polyketides and predominantly the work relating to the ethylmalonyl-CoA pathway in Rhodobacter sphaeroides. In this pathway, reductive carboxylation of (E)-crotonylCoA by a crotonyl-CoA carboxylase/reductase (Ccr) enzyme [30] with use of the purified enzyme in vitro has been described.…”

Carboxylases in Natural and Synthetic Microbial Pathways

“…Prominent examples are methylmalonyl-CoA, which is supplied by propionyl-CoA carboxylase, and ethylmalonyl-CoA, which is provided by the recently discovered CCR (28,41). A homolog of the latter enzyme was also shown to be responsible for the synthesis of the unusual extender units chloroethylmalonyl-CoA (from chlorocrotonyl-CoA) and propylmalonyl-CoA (from 2-pentenylCoA) (46,80). Moreover, in the biosynthesis of the proteasome inhibitor cinnabaramide A and the macrolide antibiotic filipin, CCR homologs supply hexylmalonyl-CoA via reductive carboxylation of 2-octenoyl-CoA (97,112,130).…”

Characterization of 2-Octenoyl-CoA Carboxylase/Reductase UtilizingpteBfromStreptomyce avermitilis

“…SalG also possesses relaxed substrate specificity toward crotonylCoA, 2-pentenyl-CoA, and 4-chlorocrotonyl-CoA. 5,6) 2-Pentenoyl-CoA carboxylase/reductase (AllR) was proposed to play a part in FK 506 biosynthesis.…”

“…4) Biochemical characterization of the CCR route in polyketide biosynthesis was first reported for the salinosporamide biosynthesis of Salinospora tropica. 5,6) SalG catalyzes the formation of ethylmalonyl-CoA, propylmalonyl-CoA, and chloroethylmalonyl-CoA to support the production of salinosporamide derivatives. The metabolic significance of SalG might lie in its production of chloroethylmalonyl-CoA.…”