Kevin D. Walker Research Interests continued
Phe Aminomutase: The first step in the construction of the N-benzoyl phenylisoserinoyl side chain attached at the C13 position of the anticancer drug Taxol produced by yew species is considered to be the conversion of 2S-α-phenylalanine to 3R-β-phenylalanine catalyzed by phenylalanine aminomutase (PAM). PAM was acquired from a Taxus cuspidata cell cDNA library, functionally expressed in Escherichia coli, and found to catalyze predominantly the vicinal isomerizaton of 2S-α- phenylalanine to 3R-β-phenylalanine; it also converts both 3S-β- (unnatural product) and 3R-β-phenylalanine to 2S-α-phenylalanine. Further studies on PAM include investigating the substrate selectivity and kinetics, assessing additional cryptic stereochemistry, surveying stable isotope exchange, and conducting structure/function assignments based on homology and X-ray crystallographic data.
Acyltransferases:
The primary objective of this project is to employ directed mutagenesis on a family of Taxus-derived acyltransferases to identify and characterize molecular determinants within the active sites that are putatively critical for catalysis and regio- and substrate specificity. A conserved motif, Cys, His, and Asp, is postulated to be involved in acyl group transfer to the acceptor molecule. By site-directed mutagenesis, we are currently assessing the critical function of the residues in this triad in relation to acyl/aroyl transfer. Defining the function of the transferases will also be accomplished through DNA-shuffling technology, and the resultant hybrid enzymes will be characterized ultimately by X-ray crystallographic analysis and molecular modeling.
Biosynthesis of Neoclerodane Diterpenoids.
Neoclerodanes are a diverse class of diterpenoid compounds of which many have demonstrated bioactivity. Despite the structural variability and differences in oxygenation and acylation, each is likely derived from a common kolavenol precursor. Evolutionarily, it is apparent that each plant making a neoclerodane has recruited a unique series of enzymes for constructing its target molecule. However, it is clear that some enzymes in these divergent pathways are similar; therefore, it is intriguing to assess if, for example, gene orthologs from Salvia divinorum, that makes κ-opioid receptor agonist salvinorin A (1), encode protein that catalyze novel conversion of metabolites occurring in the pathway to compound 3.
Accessibility of plant material provides a means to acquire the genes and characterize the corresponding enzymes on the biosynthetic pathway of the relatively structurally simpler neoclerodane salvinorin A. This effort will facilitate the identification of functional gene orthologs on the pathways of related but structurally-varied bioactive neoclerodane metabolites.
The graduate student and postdoctorate will embark on studies involving molecular cloning techniques, expression of various metabolite pathway enzymes, assay development, organic synthesis methods, basic biochemical applications and molecular biological approaches related to enzyme kinetic analyses, enzyme purification and characterization, and various spectroscopic techniques.