(582bm) Generation of Resveratrol Analogues By Enzymatic Oligomerization and Protein Engineering

Most natural polyphenols have low antimicrobial activity. In this work, we tried to generate analogues of resveratrol via either enzymatic oligomerization or site-directed mutagenesis of stilbene synthase (STS).  Enzymatic oligomerization produced two oligomers, resveratrol-trans-dihydrodimer and Pallidol. The trans-dihydrodimer showed commercially relevant antimicrobial activity against gram-positive bacteria, B. cereus (MIC = 15.0 µM), Listeria (125.0 µM), and S. aureus (MIC = 62.0 µM). Further, we identified it’s mechanism of action to be via altering membrane structure and polarization, and inhibiting DNA synthesis. This was deciphered by transcriptomic, biochemical and flow cytometric analysis. Gene expression microarray and qRT-PCR data from B. cereus indicated down regulation of ABC transporters (ATP binding proteins). Flow cytometry analysis showed depolarization of cell membrane upon treatment with resveratrol-trans-dihydrodimer. A decrease in nucleic DNA content of B. cereus and E. coli upon incubation with the trans-dihydrodimer was also observed by flow cytometry. Further, in vitro activity of DNA gyrase was strongly inhibited by the resveratrol-trans-dihydrodimer.

Next we created 15 mutants of stilbene synthase and tried to create resveratrol analogues by feeding different substrates. Type III polyketide synthases have been studied extensively over the past few decades, but most of these structural-functional studies have been carried out on chalcone synthase (CHS) like enzymes, which carry out a C6-C2 Claisen condensation of their polyketide intermediates. Stilbene synthases share 75-90% sequence similarity with CHS and form the same tetraketide intermediate, but cyclize the product via a C1-C7 aldol condensation. This cyclization requires a thioesterase-like hydrolysis step made possible by the hydrogen bonding network (HBN) present only in STSes, involving residues S338, T132 and E192 and a water molecule. Here we studied the effect of mutating functionally important residues in STS. Similar mutations have been carried out in CHS but we predicted that new products would be formed because of the different cyclization of intermediates by STS.  Mutant G256L completely lost its ability to handle the bulky p-coumaroyl-CoA substrate while a T197G mutant was still able to produce resveratrol while forming at the same time an increased amount of a more hydrophobic compound which is yet to be identified.

Finally, we have demonstrated that the enzymatic oligomerization of resveratrol is an attractive strategy for generating effective antimicrobials with potential therapeutic application. And further we plan to study the antimicrobial activity of resveratrol analogues created by mutating the functionally important residues of STS. The mechanism of action of which can be identified by carrying out studies similar to those carried out for the resveratrol-trans­-dihydrodimer.