D-3-Phosphoglycerate dehydrogenase [PGDH] catalyzes the first step in the serine biosynthetic pathway, the oxidation of 3-phosphoglycerate to 3-phosphohydroxypyruvate with the concomitant reduction of NAD. The pathway in bacteria and lower plants has evolved an inhibitory feedback mechanism that allows serine to turn off the production of itself at this first step of the pathway. The biochemical analysis of PGDH has revealed cooperativity of binding for all ligands. Structurally, the enzyme is also unique within the family of D-2-hydroxyacid dehydrogenases in being the only member to form a tetramer versus a dimer, each subunit consisting of three domains: a nucleotide, a substrate and a regulatory domain interconnected by flexible hinge regions [Panel D].

The subunit structure of the enzyme lends itself to the hypothesis of domain movements occurring at flexible hinge regions to bring about large conformational changes between domains during the catalytic reaction or in response to an inhibitor. Pictured above in A, B and C are the native enzyme, L351W PGDH and W139G PGDH, respectively. The L351W mutant places a large aromatic at the regulatory domain interface [Panel A, labeled R]. W139G removes a large hydrophobic group from the nucleotide domain interface [Panel A, labeled N]. Both mutations result in domain movements about the flexible hinge regions. Panel D illustrates the hinge movement between the substrate and nucleotide domains in the subunit structure. The new position of the domains with respect to the native enzyme and altered contacts at the subunit interfaces results in a loss of serine binding/inhibition in L351W or decreased cooperativity in serine inhibition with reduced catalytic activity in the W139G mutant. The structural analysis of these mutations along with the biochemical data suggest that subunit communication transverses extensive contacts consisting of an interdomain beta-sheet between the regulatory domains and hydrophobic interactions between the nucleotide domains in response to ligand binding.

Many proterins which are present in cellular substructures are encoded in the nucleus, translated in the cytosol, and subsequently imported into the appropriate organelle. The mechanisms by which these processes are carried out are still poorly understood. Currently graduate student Bryan Cox is cloning, purifying and studying the crystal structures of proteins from mitochondria and glyoxysomes. Most of these studies take place at the molecular level, but organelles have now been purified from two sources. Nearly all translocatable proteins have extra amino acids at the N-terminus. This so-called presequence is removed during the translocation process, so the precursor forms must be obtained by recombinant methods. It is not known whether or not there is any common conformation present in these pre-sequences. Whether or not the pre-protein is more or less stable than the mature forms has also not been studied extensively.

Several methods are being used to look at the properties of translocatable proteins including:

• X-ray crystallography
• Circular Dichroism
• Differential Scanning Calorimetry

The aim is to investigate how these pre-sequences affect conformation and perhaps their subsequent import into organelles.

So far, five translocatable proteins have been placed into expression vectors:

• Watermelon Glyoxysomal Malate Dehydrogenase
• Watermelon Mitochondrial Malate Dehydrogenase
• Rat Mitochondrial Malate Dehydrogenase
• Yeast Mitochondrial Fumarase
• Yeast Mitochondrial Isocitrate Dehydrogenase

Purification from E. coli is complicated by the presence of proteases and methods are being devised to circumvent the problem.

In order to understand the mechanism of import we have also begun to study Pex7p from Arabidopsis Thaliana. Pex7p is a cytosolic receptor for peroxisomal/glyoxysomal proteins that bind to the peroxisomal targeting signal 2 consensus sequence

(R/K)(L/V/I)X ₅ (H/Q)(L/A)

We will be looking at the binding of peroxisomal proteins to this receptor.