O'Gara M J, Lee C H, Weinberg G A, Nott J M, Queener S F
Department of Pharmacology & Toxicology, Indiana University School of Medicine, Indianapolis 46202, USA.
Antimicrob Agents Chemother. 1997 Jan;41(1):40-8. doi: 10.1128/AAC.41.1.40.
Mycophenolic acid, a specific inhibitor of IMP dehydrogenase (IMPDH; EC 1.1.1.205), is a potent inhibitor of Pneumocystis carinii growth in culture, suggesting that IMPDH may be a sensitive target for chemotherapy in this organism. The IMPDH gene was cloned as a first step to characterizing the enzyme and developing selective inhibitors. A 1.3-kb fragment containing a portion of the P. carinii IMPDH gene was amplified by PCR with two degenerate oligonucleotides based on conserved sequences in IMPDH from humans and four different microorganisms. Northern hybridization analysis showed the P. carinii IMPDH mRNA to be approximately 1.6 kb. The entire cDNA encoding P. carinii IMPDH was isolated and cloned. The deduced amino acid sequence of P. carinii IMPDH shared homology with bacterial (31 to 38%), protozoal (48 to 59%), mammalian (60 to 62%), and fungal (62%) IMPDH enzymes. The IMPDH cDNA was expressed by using a T7 expression system in an IMPDH-deficient strain of Escherichia coli (strain S phi 1101). E. coli S phi 1101 cells containing the P. carinii IMPDH gene were able to grow on medium lacking guanine, implying that the protein expressed in vivo was functional. Extracts of these E. coli cells contained IMPDH activity that had an apparent Km for IMP of 21.7 +/- 0.3 microM and an apparent Km for NAD of 314 +/- 84 microM (mean +/- standard error of the mean; n = 3), and the activity was inhibited by mycophenolic acid (50% inhibitory concentration, 24 microM; n = 2).
霉酚酸是肌苷一磷酸脱氢酶（IMPDH；EC 1.1.1.205）的特异性抑制剂，是体外培养中卡氏肺孢子虫生长的强效抑制剂，这表明IMPDH可能是该病原体化疗的敏感靶点。克隆IMPDH基因是对该酶进行特性鉴定并开发选择性抑制剂的第一步。基于人类和四种不同微生物的IMPDH保守序列，用两个简并寡核苷酸通过PCR扩增出一个1.3kb的片段，该片段包含卡氏肺孢子虫IMPDH基因的一部分。Northern杂交分析显示，卡氏肺孢子虫IMPDH mRNA约为1.6kb。分离并克隆了编码卡氏肺孢子虫IMPDH的完整cDNA。推导的卡氏肺孢子虫IMPDH氨基酸序列与细菌（31%至38%）、原生动物（48%至59%）、哺乳动物（60%至62%）和真菌（62%）的IMPDH酶具有同源性。利用T7表达系统在大肠杆菌IMPDH缺陷菌株（菌株S phi 1101）中表达IMPDH cDNA。含有卡氏肺孢子虫IMPDH基因的大肠杆菌S phi 1101细胞能够在缺乏鸟嘌呤的培养基上生长，这意味着体内表达的蛋白质具有功能。这些大肠杆菌细胞提取物含有IMPDH活性，其对肌苷一磷酸的表观Km为21.7±0.3μM，对烟酰胺腺嘌呤二核苷酸的表观Km为314±84μM（平均值±平均标准误差；n = 3），并且该活性被霉酚酸抑制（50%抑制浓度，24μM；n = 2）。
