Reungprapavut Sutarnthip, Krungkrai Sudaratana R, Krungkrai Jerapan
Unit of Biochemistry, Department of Medical Science, Faculty of Science, Rangsit University, Paholyothin Rd., Patumthani 12000, Thailand.
J Enzyme Inhib Med Chem. 2004 Jun;19(3):249-56. doi: 10.1080/14756360410001689577.
Plasmodiumfalciparum is responsible for the majority of life-threatening cases of human malaria. The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. Carbonic anhydrase (CA) is present at high levels in human red cells and in P. falciparum. Existence of at least three isozymes of the alpha < class was demonstrated in P. falciparum and a rodent malarial parasite Plasmodium berghei. The major isozyme CA1 was purified and partially characterized from P. falciparum (PfCA1). A search of the malarial genome database yielded an open reading frame similar to the alpha-CAs from various organisms, including human. The primary amino acid sequence of the PfCA1 has 60% identity with a rodent parasite Plasmodium yoelii enzyme (PyCA). The single open reading frames encoded 235 and 252 amino acid proteins for PfCA1 and PyCA, respectively. The highly conserved active site residues were also found among organisms having alpha-CAs. The PfCA1 gene was cloned, sequenced and expressed in Escherichia coli. The purified recombinant PfCA1 enzyme was catalytically active. It was sensitive to acetazolamide and sulfanilamide inhibition. Kinetic properties of the recombinant PfCA1 revealed the authenticity to the wild type enzyme purified from P. falciparum in vitro culture. Furthermore, the PfCA1 inhibitors acetazolamide and sulfanilamide showed good antimalarial effect on the in vitro growth of P. falciparum. Our molecular tools developed for the recombinant enzyme expression will be useful for developing potential antimalarials directed at P. falciparum carbonic anhydrase.
恶性疟原虫是导致大多数危及生命的人类疟疾病例的病原体。全球耐药疟原虫的出现使得鉴定和表征新型药物靶点成为必要。碳酸酐酶（CA）在人类红细胞和恶性疟原虫中大量存在。在恶性疟原虫和啮齿类疟原虫伯氏疟原虫中已证明存在至少三种α类同工酶。主要的同工酶CA1已从恶性疟原虫（PfCA1）中纯化并进行了部分表征。对疟原虫基因组数据库的搜索产生了一个与包括人类在内的各种生物体的α - CA相似的开放阅读框。PfCA1的一级氨基酸序列与啮齿类寄生虫约氏疟原虫的酶（PyCA）有60%的同一性。单个开放阅读框分别为PfCA1和PyCA编码了235和252个氨基酸的蛋白质。在具有α - CA的生物体中也发现了高度保守的活性位点残基。PfCA1基因被克隆、测序并在大肠杆菌中表达。纯化的重组PfCA1酶具有催化活性。它对乙酰唑胺和磺胺类药物抑制敏感。重组PfCA1的动力学特性显示其与从恶性疟原虫体外培养物中纯化的野生型酶具有一致性。此外，PfCA1抑制剂乙酰唑胺和磺胺类药物对恶性疟原虫的体外生长显示出良好的抗疟效果。我们为重组酶表达开发的分子工具将有助于开发针对恶性疟原虫碳酸酐酶的潜在抗疟药物。
