|Crawford, J - JOHNS HOPKINS UNIV|
|Vagstad, A - JOHNS HOPKINS UNIV|
|Townsend, C - JOHNS HOPKINS UNIV|
Submitted to: Bioorganic Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 15, 2007
Publication Date: January 12, 2008
Citation: Crawford, J.M., Vagstad, A.L., Ehrlich, K., Townsend, C.A. 2008. Starter unit specificity directs genome mining of polyketide synthase pathways in fungi. Bioorganic Chemistry. 36:16-22. Interpretive Summary: Aflatoxin production begins with the transfer of a small fatty acid to an enzyme (polyketide synthase) that makes the first stable molecule. In previous studies, we found that a special portion of the polyketide synthase is required for its ability to accept the small fatty acid. In the course of that research, we discovered that other fungi that are not known to make aflatoxin may also have a polyketide synthase that must accept a fatty acid in order for them to function. Among these fungi is the human pathogen Coccidioides immitis, which is the cause of Valley Fever in farmers in California and Arizona. After testing if the C. immitis polyketide synthase with unknown function can accept a small fatty acid, we found that, indeed, this enzyme not only accepts a fatty acid, but prefers a bigger one than the one accepted by the aflatoxin-producing polyketide synthase. We do not know what molecule is made by C. immitis, but with this knowledge we suspect that the molecule shares some relationship to aflatoxin and may be partially responsible for the disease symptoms of Valley Fever.
Technical Abstract: Search of the protein database with the aflatoxin pathway polyketide synthase (PKS) revealed putative PKSs in the pathogenic fungi Coccidioides immitis and Coccidioides posadasii that could require partnerships with a pair of fatty acid synthase (FAS) subunits for the biosynthesis of fatty acid-polyketide hybrid metabolites. A starter unit:acyl-carrier protein transacylase (SAT) domain was discovered in the nonreducing PKS. This domain is thought to accept the fatty acid product from the FAS to initiate polyketide synthesis. We expressed the C. immitis SAT domain in E. coli and showed that this domain, unlike that from the aflatoxin pathway PKS, transferred octanoyl-CoA four times faster than hexanoyl-CoA. The SAT domain also formed a covalent octanoyl intermediate and transferred this group to a free-standing ACP domain. Our results suggest that C. immitis/posadasii, both human fungal pathogens, contain a FAS/PKS cluster with functional similarity to the aflatoxin cluster found in Aspergillus species. Dissection of the PKS and determination of in vitro SAT domain specificity provides a tool to uncover the growing number of similar sequenced pathways in fungi and guide elucidation of the fatty acid-polyketide hybrid metabolites that they produce.