(528f) Protein Engineering for the Generation of Resveratrol Analogues

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.  Interestingly a single mutation, S338V, converted the STS to CHS possibly due to the disruption of the HBN. 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.  We plan to further feed 15 other mutants created with different substrates and attempt to identify products formed.