Robert Shepherd

My research is focused on the development of analytical workflows to assess the activity of complex enzymatic reactions. Specifically, I am using trapped ion mobility spectrometry to study the relative abundance of isomeric neuroactive molecules produced by genetically engineered enzymes.

Kainoid synthases (KaSs) are a small subset of the Fe/ɑKG family of enzymes that natively catalyze cyclization via selective C H activation, rather than the usual hydroxylation activity observed for most other Fe/ɑKG enzymes. KabC from the red macroalgae Digenea simplex catalyzes the cyclization of a prenylated L-glutamic acid substrate, prekainic acid (PKA), resulting in the formation of kainic acid (KA), a potent human ionotropic glutamate receptor (iGluR) agonist, and a minor product, kainic acid lactone (KAL), which has shown antagonistic activity at iGluRs. Notably, a KabC ortholog from Grateloupia filicina facilitates the production of primarily KAL, highlighting the catalytic promiscuity of the KabC enzymes. However, GfKabC does not express well in E. coli and is difficult to purify, making direct access to KAL difficult using current biosynthetic methods. Implementing novel analytical technology, such as trapped ion mobility spectrometry (TIMS), to rapidly screen for Kab enzyme activity will allow us to more rapidly expand access to diverse neuroactive tools that are difficult to obtain using conventional synthetic techniques.