Hydrolase involved in brefeldin A biosynthesis controls chain length of polyketide
Monday, April 28, 2014
Exhibit/Poster Hall, lower level (Hilton Clearwater Beach)
Angelica Zabala and Yi Tang, Chemical and Biomolecular Engineering, University of California - Los Angeles, Los Angeles, CA
Brefeldin A is a natural product from the filamentous fungi Eupenicillium brefeldianum with a wide-range of bioactivities, including antiviral, anti fungal and cytotoxic against several cancer cell lines. However, it is most known for its inhibition of protein transport among eukaryotes, which makes it a useful tool in biological labs. This compound is a 16-membered macrolactone that contains an unusual cyclopentane ring. By sequencing the producing strain and performing bioinformatic analysis on the assembled genome, we have identified the biosynthetic cluster that includes a highly-reducing polyketide synthase (HR-PKS, herein referred to as BrefA), a hydrolase (BrefB) and several P450 monooxygenases. We investigated the activity of the megasynthase BrefA and the releasing enzyme BrefB both in vitro and in vivo (in a Saccharomyces cerevisiae host), and found that they produce four linear octaketide molecules differing in the degree of reduction of the polyketide scaffold, which substantiates the involvement of the PKS in creating the C-16 backbone of the molecule. Interestingly, base hydrolysis or use of a non-cognate hydrolase releases a longer-chained product, implicating that the hydrolase BrefB controls the chain-length of the polyketide. Mutation of its active site residues renders the enzyme unable to strictly regulate the number of extension. This study demonstrates the significance of the accessory enzyme in determining the chain length of the polyketide product. This is contrary to what was previously believed that the programming rules lie on the PKS and its domains.