Protease trafficking in two primitive eukaryotes is mediated by a prodomain protein motif.

Trypanosome protozoa, an early lineage of eukaryotic cells, have proteases homologous to mammalian lysosomal cathepsins, but the precursor proteins lack mannose 6-phosphate. Utilizing green fluorescent protein as a reporter, we demonstrate that the carbohydrate-free prodomain of a trypanosome cathepsin L is necessary and sufficient for directing green fluorescent protein to the lysosome/endosome compartment. A proper prodomain/catalytic domain processing site sequence is also required to free the mature protease for delivery to the lysosome/endosome compartment. A nine-amino acid prodomain loop motif, implicated in prodomain-receptor interactions in mammalian cells, is conserved in the protozoa. Site-directed mutagenesis now confirms the importance of this loop to protease trafficking and suggests that a protein motif targeting signal for lysosomal proteases arose early in eukaryotic cell evolution.

Crystal structure of Tritrichomonas foetus inosine-5'-monophosphate dehydrogenase and the enzyme-product complex.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is an attractive drug target for the control of parasitic infections. The enzyme catalyzes the oxidation of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), the committed step in de novo guanosine monophosphate (GMP) biosynthesis. We have determined the crystal structures of IMPDH from the protozoan parasite Tritrichomonas foetus in the apo form at 2.3 A resolution and the enzyme-XMP complex at 2.6 A resolution. Each monomer of this tetrameric enzyme is comprised of two domains, the largest of which includes an eight-stranded parallel beta/alpha-barrel that contains the enzyme active site at the C termini of the barrel beta-strands. A second domain, comprised of residues 102-220, is disordered in the crystal. IMPDH is expected to be active as a tetramer, since the active site cavity is formed by strands from adjacent subunits. An intrasubunit disulfide bond, seen in the crystal structure, may stabilize the protein in a less active form, as high concentrations of reducing agent have been shown to increase enzyme activity. Disorder at the active site suggests that a high degree of flexibility may be inherent in the catalytic function of IMPDH. Unlike IMPDH from other species, the T. foetus enzyme has a single arginine that is largely responsible for coordinating the substrate phosphate in the active site. This structural uniqueness may facilitate structure-based identification and design of compounds that specifically inhibit the parasite enzyme.

Identification of the IMP binding site in the IMP dehydrogenase from Tritrichomonas foetus.

The IMP dehydrogenase from Tritrichomonas foetus has been identified as a potential target for antitritrichomonial chemotherapy. The gene encoding this enzyme was expressed in transformed Escherichia coli, and the recombinant protein was purified to homogeneity with an average yield of 3 mg of protein per liter of bacterial culture. Kinetic characterizations verified that the recombinant enzyme is in the authentic native state. 6-Cl-IMP, an irreversible inhibitor of the enzyme, was found to protect cysteine residue 319 of the enzyme against carboxymethylation by iodoacetamide. Radiolabeled IMP was covalently bound to the enzyme during the enzyme-catalyzed reaction via the formation of a specific adduct with cysteine residue 319. It is thus postulated that the conversion of IMP to XMP catalyzed by the IMP dehydrogenase from T. foetus is mediated by a nucleophilic attack of cysteine-319 in the enzyme protein to IMP at, most likely, its 2-position to facilitate a hydride transfer to NAD, resulting in the formation of a covalent intermediate between substrate and enzyme.