Kyle Price and Samuel Tarman, “Investigations of AEQ67_06425 and AEQ67_06430, Enzymes of Unknown Function from Pseudomonas sp. RIT-PI-q”
Mentor: Nicholas Silvaggi, Chemistry & Biochemistry
Poster #177
The Gram-negative bacterium Pseudomonas sp. RIT-PI-q RW1 was isolated in Rochester, NY from poison ivy internal stem tissue. The genome of this organism contains a pair of overlapped, likely co-expressed genes, AEQ67_06425 and AEQ67_06430. The product of the AEQ67_06430 appears to be an enzyme of unknown function with significant sequence identity to acetoacetate decarboxylase, placing it in the acetoacetate decarboxylase-like superfamily (ADCSF). We refer to this protein as PsADC. The ADCSF is a poorly characterized group of enzymes whose reaction and substrate specificities are not well known, though differences in active site residues among ADCSF members suggest that there is significant diversity in both substrate and reaction specificities. PsADC is a small (29.3 kDa), tetrameric ADCSF enzyme that, unlike the prototypical members of the superfamily, does not have acetoacetate decarboxylase activity. The second gene, AEQ67_06425, codes for an enzyme annotated as a tautomerase. Our structural characterization shows that the tertiary structure of this protein is similar to malonate semialdehyde dehydrogenase, and so we refer to this protein as PsMSAD. Our preliminary steady state kinetics data show that, while it is clearly not the biologically-relevant substrate, PsADC catalyzes the condensation of pyruvate and cinnamaldehyde to yield a dienone product that could be called cinnamylidenepyruvate. We hypothesize that it may also be able to catalyze a retro-aldol cleavage of 2-oxo-3-hexenedioate to give pyruvate and malonate semialdehyde, though this is still being investigated. The activity of the tautomerase homolog PsMSAD is less clear. The data presented here cover the cloning and expression of a SUMO-tagged version of PsADC together with its crystallization and preliminary steady state kinetics analysis. Crystals of PsOT were grown in an attempt to trap potential enzyme-substrate complex structures that might give clues into the substrate specificity and/or possible reaction catalyzed by PsMSAD.