DataSheet_1_The Structural Basis of Babesia orientalis Lactate Dehydrogenase.docx

Glycolytic enzymes play a crucial role in the anaerobic glycolysis of apicomplexan parasites for energy generation. Consequently, they are considered as potential targets for new drug development. Previous studies revealed that lactate dehydrogenase (LDH), a glycolytic enzyme, is a potential drug target in different parasites, such as Plasmodium, Toxoplasma, Cryptosporidium, and Piroplasma. Herein, in order to investigate the structural basis of LDH in Babesia spp., we determined the crystal structure of apo Babesia orientalis (Bo) LDH at 2.67-Å resolution in the space group P1. A five-peptide insertion appears in the active pocket loop of BoLDH to create a larger catalytic pocket, like other protozoa (except for Babesia microti LDH) and unlike its mammalian counterparts, and the absence of this extra insertion inactivates BoLDH. Without ligands, the apo BoLDH takes R-state (relaxed) with the active-site loop open. This feature is obviously different from that of allosteric LDHs in T-state (tense) with the active-site loop open. Compared with allosteric LDHs, the extra salt bridges and hydrogen bonds make the subunit interfaces of BoLDH more stable, and that results in the absence of T-state. Interestingly, BoLDH differs significantly from BmLDH, as it exhibits the ability to adapt quickly to the synthetic co-factor APAD+. In addition, the enzymatic activity of BoLDH was inhibited non-competitively by polyphenolic gossypol with a Ki value of 4.25 μM, indicating that BoLDH is sensitive to the inhibition of gossypol and possibly to its new derivative compounds. The current work provides the structural basis of BoLDH for the first time and suggests further investigation on the LDH structure of other Babesia spp. That knowledge would indeed facilitate the screening and designing of new LDH inhibitors to control the intracellular proliferation of Babesia spp.

糖酵解酶（Glycolytic enzymes）在顶复门寄生虫的无氧糖酵解能量产生过程中发挥关键作用。因此，它们被视为新型药物开发的潜在靶点。既往研究表明，乳酸脱氢酶（lactate dehydrogenase, LDH）作为一种糖酵解酶，是疟原虫、弓形虫、隐孢子虫以及梨形虫等多种寄生虫的潜在药物靶点。本研究为探究巴贝斯虫属（Babesia spp.）LDH的结构基础，解析了脱辅基东方巴贝斯虫（Babesia orientalis, Bo）LDH的晶体结构，分辨率达2.67 Å，空间群为P1。与哺乳动物同源蛋白不同，且与除微小巴贝斯虫LDH外的其他原生动物一致，BoLDH的活性口袋环存在一段五肽插入序列，可形成更大的催化口袋；若缺失这段额外插入序列，则会使BoLDH失活。在无配体状态下，脱辅基BoLDH以松弛态（R态）存在，其活性位点环处于开放状态。这一特征与处于紧张态（T态）且活性位点环开放的别构LDH显著不同。相较于别构LDH，BoLDH的亚基界面存在额外的盐桥与氢键，使其更为稳定，这也导致其不存在紧张态（T态）。有趣的是，BoLDH与微小巴贝斯虫LDH（BmLDH）存在显著差异，它能够快速适配人工合成的辅助因子APAD+。此外，多酚类棉酚可通过非竞争性抑制方式抑制BoLDH的酶活性，其抑制常数（Ki）为4.25 μM，表明BoLDH对棉酚及其潜在新型衍生物的抑制作用敏感。本研究首次阐明了BoLDH的结构基础，并建议对其他巴贝斯虫属物种的LDH结构开展进一步研究。相关研究成果将有助于筛选并设计新型LDH抑制剂，从而抑制巴贝斯虫属原虫的细胞内增殖。