Organelle enzymes encoded by nuclear genes are often targeted and translocated using extra N-terminal residues. Little is known regarding the effect of these additional amino acids on the conformation of the protein. Several different attempts to isolate and crystallize the recombinant pre-mitochondrial malate dehydrogenase from rat liver were thwarted by the presence of E. coli proteases. The successful purification of an organelle precursor was demonstrated by Gietl and co-workers (Gietl et. al., 1996) who showed that the pre-glyoxysomal malate dehydrogenase (pre-gMDH) from watermelon is stable in E. coli extracts. They isolated large amounts of pre-gMDH by preparing the expression system with a histidine arm and then using a Nickel affinity column for purification. Using the Gietl constructs, both the precursor and mature forms of watermelon gMDH have been purified. Crystals and x-ray data for the mature form have been obtained. Amino acid sequence analyses of pre-gMDH show that only a small fraction of the precursor has been cleaved 32 residues from the N-terminal end. To reduce proteolysis, the purification protocol of pre-gMDH includes the addition of protease inhibitors in all buffers and an additional cation exchange column for removing trace protein contaminants. Small crystalline specimens of pre-gMDH have been obtained in the presence of 20% PEG 8000 and 0.2M magnesium acetate in sodium cacodylate buffer at pH 6.5 using hanging drop vapor diffusion methods.
In plants, there are isoforms of malate dehydrogenase (MDH) in different compartments of the cells which participate in different metabolic pathways (Gietl, 1992). The isoenzymes are nuclear-encoded and synthesized in the cytosol. The organelle isoenzymes such as mitochondrial and glyoxysomal MDHs are synthesized as precursors with N-terminal presequences, which are cleaved off upon import into their organelles (Hartl et. al., 1988).
Gietl, et. al. (1990a, 1990b) isolated and cloned the genes encoding the precursor forms of glyoxysomal and mitochondrial malate dehydrogenases from watermelon (p-gMDH and p-mMDH, respectively). p-gMDH has a 37-residue long presequence and a 320-residue long mature sequence. No sequence homology is found in their presequences, which target the precursor proteins into their respective organelles. The presequence of p-gMDH contains the peroxisomal targeting signal RL/I-X5-HL (Gietl, et. al., 1996). That of p-mMDH contains the features of mitochondrial targeting sequences: hydrophilic and positively charged (Gietl et. al, 1996; Hartl, et. al., 1988). The mature sequences of the precursor proteins are 65% identical and 76% similar. Both p-gMDH and p-mMDH have been found to be enzymatically active.
Although many studies have been done on organelle import systems, especially those imported to mitochondria, little is known about the conformation of a precursor protein prior to or during translocation into its organelle. This study focuses on crystallographic analysis of p-gMDH and gMDH. A comparison of the crystal structures of p-gMDH and gMDH will give us insights into the structural effect of the presequence on the mature conformation and perhaps potential mechanisms for membrane translocation.