The Lactate/Malate Dehydrogenase family of enzymes are conserved enzymes that play important roles in carbon metabolism in a variety of organisms. This family also contains a less well-characterized 2-Hydroxyisocaproate Dehydrogenase which is found in some bacteria, mainly those associated with dairy product fermentation. I have been developing a pair of courses that will begin using these proteins as a model system to investigate substrate specificity in Course-base Undergradaute Research Experience (CURE). The first course focuses on computational techniques to have students identify differences in sequence conservation and model protein/substrate interactions. The first time offering this course provided preliminary data predicting uncharacterized hydroxyisocaproate dehydrogenase enzymes are likely to have promiscuous lactate activity, and identifying residues that might interfere with lactate binding to a malate dehydrogenase enzyme. Results like this will be used in the second course teaching experimental biochemical techniques while introducing mutations to test enzyme specificity. While this course will be taught as a CURE for the first time in fall of 2024, during independent projects that were part of the old course model students have successfully cloned the lacatate dehydrogenase and hydroxyisocaproate dehydrogenase from the same strain of Lacticaseibaciilus paracasei into expression vectors and initial purification and characterization studies are currently underway. These will be used as model systems along with a previously cloned malate dehydrogenase from the malaria parasite Plasmodium falciparum.

The three reactions that are being studied as part of this reaction and an example sequence conservation logo and substrate binding model

Haley Colling

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