Characterizing the Kinetic Behavior of Candidate Threonine Transaldolases

Enzymes are appealing catalysts, driving specific chemical reactions under ambient conditions in aqueous environments. Upon incorporation into protein sequences, non-standard amino acids (nsAAs) can imbue enzymes with novel chemistry, unlocking avenues for biocatalysis and pharmaceutical applications. Not only can nsAAs greatly contribute to the development of innovative protein technologies, but some nsAAs have pharmacological relevance themselves. Threonine transaldolases (TTAs), which comprise a recently characterized enzyme class, condense an aldehyde with threonine to synthesize nsAAs with β-hydroxy groups. These enzymes have great potential for β-hydroxy-nsAA synthesis given their high enantioselectivity and thermodynamic favorability in biocatalysis, but only one TTA (ObaG from Pseudomonas fluorescens) has been studied closely in literature. My research seeks to characterize the substrate specificity and kinetic behavior of other candidate TTAs. Using a sequence similarity network constructed around ObaG, our group selected eight sequences to probe a diverse spread of potential TTA chemistries. Through in vitro analysis with a coupled-enzyme assay, I verified that one of the TTA enzyme candidates I cloned, expressed, and purified demonstrates TTA chemistry. Compared to ObaG and three other TTAs previously-confirmed by our group, this new TTA has the lowest K_M for threonine when using the model substrate phenylacetaldehyde. This finding indicates that the new TTA has an enhanced affinity for its threonine donor—a metric initially reported as a hurdle for ObaG kinetics.