Kevin D. Walker Research Interests
Functional Analysis of Enzymes on Biosynthetic Pathways of Plant-derived Bioactive Compounds
Our research uses a multidisciplinary approach to elucidate biosynthetic pathways of secondary metabolites that have a potentially beneficial biological effect. Acquisition and characterization of the genes, and corresponding gene products, on various pathways to bioactive compounds provides the field of natural products biochemistry with tools for potential application in the biotransformation of natural products or synthetically-derived chemicals that are non-native substrates of the isolated enzymes. These functionally-defined genes can also be bioengineered into a suitable host organism to potentially alter the metabolic profile of interesting molecules in vivo.
Taxol pathway enzymes: mechanism and selectivity.
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.
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.
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