Cloning, sequencing, and structural analysis of the DNA encoding inosine monophosphate dehydrogenase (EC 1.1.1.205) from Tritrichomonas foetus.
The inosine monophosphate dehydrogenase (IMPDH) of the parasitic protozoan Tritrichomonas foetus is a purine salvage enzyme with a subunit molecular weight of 58,000. The enzyme has been purified to homogeneity by Verham et al. (Molecular and Biochemical Parasitology 24, 1-12, 1987) and characterized in more detail by Hedstrom and Wang (Biochemistry 29, 849-854, 1990). We used a polyclonal antibody directed against the purified enzyme to identify three cDNA clones from T. foetus. These clones were sequenced and found to contain an open reading frame encoding 497 amino acids. By complementation studies on an Escherichia coli mutant with its IMPDH gene deleted, the cDNA clones were able to transform the bacterial cells to grow on minimal medium without guanine. One of the cDNA clones, 2aa1, was used to identify two genomic clones, 2d1c and 3m4b, both containing a 4.1-kb HindIII fragment. The fragment was subcloned into the Bluescript KS+ plasmid, sequenced, and found to contain the same open reading frame as the cDNA clone except that it encodes six additional amino acid residues at the N-terminus. Its sequence has a 34% identity with that of the human IMPDH, 32% with that of E. coli IMPDH, and 31% with that of Leishmania donovani IMPDH. The molecular weight of the deduced protein is 55,534. Two segments of polypeptide that are conserved in all other IMPDHs, containing the putative NAD+ and IMP binding sites, are also relatively conserved in T. foetus. Since the parasite enzyme differs from the bacterial and mammalian IMPDHs by a very high Km value for NAD+ and an even higher KI value for mycophenolic acid (MPA) (Verham et al. 1987; Hedstrom and Wang 1990), the sequence of the parasite enzyme may provide information on the mechanism of MPA binding and the chance for other specific inhibitor design.
Beck
J T
JT
Department of Pharmaceutical Chemistry, University of California at San Francisco 94143.
Zhao
S
S
Wang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017124:Conserved Sequence; D016054:DNA, Protozoan / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D007168:IMP Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000737:chemistry; D016415:Sequence Alignment; D016835:Tritrichomonas foetus / Q000201:enzymology* / Q000235:genetics
D016054:DNA, Protozoan; D012333:RNA, Messenger; D007168:IMP Dehydrogenase