Enzymatic Properties and Biological Roles of Novel Beta-Hydroxyacid Dehydrogenases in Plants and Bacteria

Indiana University, Bloomington IN

Investigators

Abstract

The beta-hydroxyacid dehydrogenases are a structurally and mechanistically related family of enzymes with a predominance of homologues specific to bacteria and plants. The only two known mammalian family members include beta-hydroxyisobutyrate dehydrogenase and 6-phosphogluconate dehydrogenase. Two of the bacterial family members have recently been identified as isozymes of tartronate semialdehyde reductase (TSR) with differences in substrate specificity. TSR is part of a pathway for glycerate biosynthesis from glyoxylate, hydroxypyruvate, and other substrates in bacteria and plants. Most bacterial and plant genomes display multiple homologues of TSR. The Escherichia coli genome contains 4 homologues, of which two have been cloned and enzymatically characterized. The Arabidopsis thaliana genome contains 6 homologues of TSR, none of which have been cloned or identified in terms of substrate specificity and enzymatic properties. Most of the bacterial beta-hydroxyacid dehydrogenase genes contain apparent binding sites for cAMP-CRP and are likely regulated by catabolite repression. These enzymes may serve alternative metabolic pathways, such as glycerate biosynthesis, in anaerobiosis and in conditions of nutrient deprivation when TCA cycle activity is inadequate. This hypothesis is fitting with the organization of surrounding genes and cAMP-CRP sites observed in E. coli and Haemophilus influenzae loci containing beta-hydroxyacid dehydrogenase genes. This project will examine the enzymatic properties and substrate specificities of cloned and over-expressed beta-hydroxyacid dehydrogenases of A. thaliana, to determine what biological functions the plant TSR isozymes may serve. The identities and relative quantities of these and other co-expressed proteins will be determined by a proteomic approach, utilizing isotope-coded affinity tags, in order to understand more clearly what metabolic pathways are functioning together under conditions of specific substrate limitation and/or anaerobiosis in E. coli and H. influenzae. It is not clear, biologically, why certain bacteria and plants should require multiple isoforms of TSR, but this genetic capacity may reflect a previously unrecognized importance of glycerate biosynthesis in bacteria and plants. This project will expand our understanding of this recently discovered family of enzymes, and their apparently specific metabolic roles in plants and bacteria. This research will also bring new information about what enzymes and other proteins are predominantly expressed in two different bacterial species when grown under various limiting and anaerobic conditions.