Beck J T, Zhao S, Wang C C
Department of Pharmaceutical Chemistry, University of California at San Francisco 94143.
Exp Parasitol. 1994 Feb;78(1):101-12. doi: 10.1006/expr.1994.1010.
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.
寄生原生动物胎儿三毛滴虫的肌苷单磷酸脱氢酶（IMPDH）是一种亚基分子量为58,000的嘌呤补救酶。Verham等人（《分子与生化寄生虫学》24，1 - 12，1987）已将该酶纯化至同质，并由Hedstrom和Wang进行了更详细的表征（《生物化学》29，849 - 854，1990）。我们使用针对纯化酶的多克隆抗体从胎儿三毛滴虫中鉴定出三个cDNA克隆。对这些克隆进行测序，发现其包含一个编码497个氨基酸的开放阅读框。通过对缺失其IMPDH基因的大肠杆菌突变体进行互补研究，这些cDNA克隆能够使细菌细胞在不含鸟嘌呤的基本培养基上生长。其中一个cDNA克隆2aa1被用于鉴定两个基因组克隆2d1c和3m4b，二者均含有一个4.1 kb的HindIII片段。该片段被亚克隆到Bluescript KS +质粒中，进行测序，发现其与cDNA克隆含有相同的开放阅读框，只是在N端多编码了六个氨基酸残基。其序列与人类IMPDH的序列有34%的同源性，与大肠杆菌IMPDH有32%的同源性，与杜氏利什曼原虫IMPDH有31%的同源性。推导蛋白的分子量为55,534。在所有其他IMPDH中保守的、包含假定的NAD +和IMP结合位点的两段多肽，在胎儿三毛滴虫中也相对保守。由于寄生虫酶与细菌和哺乳动物的IMPDH相比，对NAD +的Km值非常高，对霉酚酸（MPA）的KI值甚至更高（Verham等人，1987；Hedstrom和Wang，1990），寄生虫酶的序列可能为MPA结合机制及其他特异性抑制剂设计提供信息。
