School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
PLoS One. 2012;7(3):e33138. doi: 10.1371/journal.pone.0033138. Epub 2012 Mar 16.
Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of an unusual amino acid hypusine (N(€)-(4-amino-2-hydroxybutyl) lysine), which is present on only one cellular protein, eukaryotic initiation factor 5A (eIF5A). We present here the molecular and structural basis of the function of DOHH from the protozoan parasite, Leishmania donovani, which causes visceral leishmaniasis. The L. donovani DOHH gene is 981 bp and encodes a putative polypeptide of 326 amino acids. DOHH is a HEAT-repeat protein with eight tandem repeats of α-helical pairs. Four conserved histidine-glutamate sequences have been identified that may act as metal coordination sites. A ~42 kDa recombinant protein with a His-tag was obtained by heterologous expression of DOHH in Escherichia coli. Purified recombinant DOHH effectively catalyzed the hydroxylation of the intermediate, eIF5A-deoxyhypusine (eIF5A-Dhp), in vitro. L. donovani DOHH (LdDOHH) showed ~40.6% sequence identity with its human homolog. The alignment of L. donovani DOHH with the human homolog shows that there are two significant insertions in the former, corresponding to the alignment positions 159-162 (four amino acid residues) and 174-183 (ten amino acid residues) which are present in the variable loop connecting the N- and C-terminal halves of the protein, the latter being present near the substrate binding site. Deletion of the ten-amino-acid-long insertion decreased LdDOHH activity to 14% of the wild type recombinant LdDOHH. Metal chelators like ciclopirox olamine (CPX) and mimosine significantly inhibited the growth of L. donovani and DOHH activity in vitro. These inhibitors were more effective against the parasite enzyme than the human enzyme. This report, for the first time, confirms the presence of a complete hypusine pathway in a kinetoplastid unlike eubacteria and archaea. The structural differences between the L. donovani DOHH and the human homolog may be exploited for structure based design of selective inhibitors against the parasite.
脱羟鸟氨酸羟化酶（DOHH）催化翻译后合成一种特殊氨基酸——hypusine（N(€)-(4-氨基-2-羟基丁基)赖氨酸）的最后一步，该氨基酸仅存在于一种细胞蛋白——真核起始因子 5A（eIF5A）上。我们在此介绍了引起内脏利什曼病的原生动物寄生虫利什曼原虫中的 DOHH 的分子和结构基础。利什曼原虫 DOHH 基因长 981bp，编码一个由 326 个氨基酸组成的假定多肽。DOHH 是一种 HEAT 重复蛋白，具有八个串联的α-螺旋对。已经鉴定出四个保守的组氨酸-谷氨酸序列，它们可能作为金属配位位点发挥作用。通过在大肠杆菌中异源表达 DOHH，获得了带有 His 标签的约 42kDa 的重组蛋白。纯化的重组 DOHH 可有效地在体外催化中间产物 eIF5A-脱羟鸟氨酸（eIF5A-Dhp）的羟化。利什曼原虫 DOHH（LdDOHH）与人同源物的序列同一性约为 40.6%。LdDOHH 与人同源物的比对显示，前者有两个明显的插入，对应于蛋白质 N 端和 C 端两半之间的可变环连接的对齐位置 159-162（四个氨基酸残基）和 174-183（十个氨基酸残基），后者存在于靠近底物结合位点附近。缺失十个氨基酸长的插入片段使 LdDOHH 的活性降低至野生型重组 LdDOHH 的 14%。金属螯合剂如环吡酮胺（CPX）和金雀异黄素在体外显著抑制利什曼原虫的生长和 DOHH 活性。这些抑制剂对寄生虫酶的抑制作用比对人源酶的抑制作用更为有效。本报告首次证实，与真细菌和古细菌不同，动基体生物中存在完整的 hypusine 途径。LdDOHH 与人类同源物之间的结构差异可能被用于针对寄生虫的基于结构的选择性抑制剂的设计。
