Brooks D R, Wang P, Read M, Watkins W M, Sims P F, Hyde J E
Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology, England.
Eur J Biochem. 1994 Sep 1;224(2):397-405. doi: 10.1111/j.1432-1033.1994.00397.x.
Dihydropteroate synthase (H2Pte synthase) is the target of the sulfur-based antimalarial drugs, which are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (H2folate reductase) to combat chloroquine-resistant malaria. We have isolated the H2Pte synthase coding sequence of the most pathogenic human parasite Plasmodium falciparum. It forms part of a longer coding sequence, located on chromosome 8, that also specifies 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (CH2OH-H2pterinPP kinase) at its 5' proximal end. This domain is unusually large, with two long insertions relative to other CH2OH-H2pterinPP kinase molecules. To investigate a possible genetic basis for clinical resistance to sulfa drugs, we sequenced the complete H2Pte synthase domains from eleven isolates of P. falciparum with diverse geographical origins and levels of sulfadoxine resistance. Overall, point mutations in five positions were observed, affecting four codons. Parasite lines exhibiting high-level resistance were found to carry either a double mutation, altering both Ser436 and Ala613, or a single mutation affecting Ala581. The mutations at positions 436 and 581 have the same location relative to each of two degenerate repeated amino acid motifs that are conserved across all other known H2Pte synthase molecules. The amino acid alteration at residue 613 is identically positioned relative to a different conserved motif. The fourth amino acid residue (437) affected by mutation, though adjacent to the apparently crucial residue 436, shows no obvious correlation with resistance. Although these mutations have no exact counterparts in any other organism, that at position 581 falls within a region of three amino acids where H2Pte synthase is modified in various ways in a number of sulfonamide-resistant pathogenic bacteria. Copy-number analysis indicated that there was no amplification of the H2Pte synthase domain in resistant parasite lines of P. falciparum, compared to sensitive lines.
二氢蝶酸合酶（H2Pte合酶）是硫基抗疟药物的作用靶点，这些药物常与二氢叶酸还原酶（H2叶酸还原酶）抑制剂联合使用，以对抗耐氯喹疟疾。我们分离出了最具致病性的人类寄生虫恶性疟原虫的H2Pte合酶编码序列。它是位于8号染色体上一个较长编码序列的一部分，该序列在其5'近端还编码6-羟甲基-7,8-二氢蝶呤焦磷酸激酶（CH2OH-H2蝶呤PP激酶）。该结构域异常大，相对于其他CH2OH-H2蝶呤PP激酶分子有两个长插入序列。为了研究对磺胺类药物临床耐药性的可能遗传基础，我们对来自不同地理来源和不同磺胺多辛耐药水平的11株恶性疟原虫的完整H2Pte合酶结构域进行了测序。总体而言，观察到五个位置的点突变，影响四个密码子。发现表现出高水平耐药性的寄生虫株携带双重突变，改变了Ser436和Ala613，或单个影响Ala581的突变。436和581位的突变相对于在所有其他已知H2Pte合酶分子中保守的两个简并重复氨基酸基序中的每一个具有相同位置。613位氨基酸的改变相对于一个不同的保守基序位置相同。受突变影响的第四个氨基酸残基（437），尽管与明显关键的残基436相邻，但与耐药性没有明显相关性。尽管这些突变在任何其他生物体中都没有完全对应的突变，但581位的突变位于一个由三个氨基酸组成的区域内，在许多耐磺胺类药物的病原菌中，H2Pte合酶在该区域以各种方式发生修饰。拷贝数分析表明，与敏感株相比，恶性疟原虫耐药寄生虫株中H2Pte合酶结构域没有扩增。
