Current CUREs using Watermelon Glyoxysomal Malate Dehydrogenase are focused around six project areas:
Active Site (Catalysis) involves residues directly or indirectly responsibe for the hydride transfer and proton abstraction steps in the overall catalysis. While clear roles for several “first sphere” catalytic residues are well established, how “second sphere” residues contribute to either catalysis of allosteric interactions are unknown.
2. Mobile Loop (Substrate Specificity etc) involves the mobile loop that closes over the active site. This loop not only controls substrate specificity (choice of 3, 4 or 5 carbon substrates) but is also thought to contribute to both catalysis and cofactor binding. While some of the factors controlling 3 carbon versus 4 carbon substrates have been explored, little is know about how the active sites accommodates the 5 carbon oncometabolite precursor alphaketoglutarate.
3. Interface (Allosteric Regulation) explores the subunit interface in the cannonical dimer structure of MDH which plays a significant role in cooperative binding and in a “reciprocating subunit” mechanism for the enzyme. This research area is starting to detail the interactions that both trigger and mediate subunit interactions.
4. Citrate Regulation is complex in Malate Dehydrogenases, involving both activation and inhibition depending on species and isoform. While only binding at the active site is reported, Citrate does not appear to be a competitive inhibitor. Crystal structures of inhibited and uninhibited forms show two major areas (both involving alpha helices) of the protein show significant conformational differences. What triggers these conformational changes and how they impact overall function is currently unknown
5. Loops & Turns (Protein Folding & Stability) explores the roles of the various loops and turns found in the protein and how they are involved in both structure acquisition (folding) and global and local stability. What are the rules that govern folding in acomplex alpha/beta protein such as Malate Dehydrogenase? What are the links between stability/flexibility and enzyme function?
6. Rossmann Fold (Cofactor Binding & Specificity). Like most dehydrogenases, Malate Dehydrogenase has a so-called Rossmann fold associated with confactor binding. Many questions remain including how the Rossmann fold is tailored to recognize NAD versus NADP in various isoforms, and which components of the Rossmann fold contribute to cofactor induced conformational changes involved in catalysis and in regulation.
Each of these areas involve many unanswered questions that can form the basis of an effective Malate Dehydrogenase CUREs Community project involving either a single class or collaboration between classes at different institutions.