Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India.
PLoS One. 2013;8(1):e53397. doi: 10.1371/journal.pone.0053397. Epub 2013 Jan 11.
Polyamine biosynthetic pathway is a validated therapeutic target for large number of infectious diseases including cancer, giardiasis and African sleeping sickness, etc. α-Difluoromethylornithine (DFMO), a potent drug used for the treatment of African sleeping sickness is an irreversible inhibitor of ornithine decarboxylase (ODC), the first rate limiting enzyme of polyamine biosynthesis. The enzyme ODC of E. histolytica (EhODC) has been reported to exhibit resistance towards DFMO.
METHODOLOGY/PRINCIPAL FINDING: The basis for insensitivity towards DFMO was investigated by structural analysis of EhODC and conformational modifications at the active site. Here, we report cloning, purification and crystal structure determination of C-terminal truncated Entamoeba histolytica ornithine decarboxylase (EhODCΔ15). Structure was determined by molecular replacement method and refined to 2.8 Å resolution. The orthorhombic crystal exhibits P2(1)2(1)2(1) symmetry with unit cell parameters a = 76.66, b = 119.28, c = 179.28 Å. Functional as well as evolutionary relations of EhODC with other ODC homologs were predicted on the basis of sequence analysis, phylogeny and structure.
CONCLUSIONS/SIGNIFICANCE: We determined the tetrameric crystal structure of EhODCΔ15, which exists as a dimer in solution. Insensitivity towards DFMO is due to substitution of key substrate binding residues in active site pocket. Additionally, a few more substitutions similar to antizyme inhibitor (AZI), a non-functional homologue of ODCs, were identified in the active site. Here, we establish the fact that EhODC sequence has conserved PLP binding residues; in contrast few substrate binding residues are mutated similar to AZI. Further sequence analysis and structural studies revealed that EhODC may represent as an evolutionary bridge between active decarboxylase and inactive AZI.
多胺生物合成途径是许多传染病的有效治疗靶点，包括癌症、贾第虫病和非洲昏睡病等。α-二氟甲基鸟氨酸（DFMO）是一种用于治疗非洲昏睡病的有效药物，是鸟氨酸脱羧酶（ODC）的不可逆抑制剂，ODC 是多胺生物合成的限速酶。已报道溶组织内阿米巴（EhODC）的 ODC 对 DFMO 具有抗性。
方法/主要发现：通过对 EhODC 的结构分析和活性部位构象修饰，研究了对 DFMO 不敏感的原因。在这里，我们报告了末端截断的溶组织内阿米巴鸟氨酸脱羧酶（EhODCΔ15）的克隆、纯化和晶体结构测定。结构通过分子置换法确定，并精修至 2.8 Å 分辨率。该正交晶体表现出 P2(1)2(1)2(1)对称，单位细胞参数为 a = 76.66、b = 119.28、c = 179.28 Å。基于序列分析、系统发育和结构，预测了 EhODC 与其他 ODC 同源物的功能和进化关系。
结论/意义：我们确定了 EhODCΔ15 的四聚体晶体结构，该结构在溶液中以二聚体形式存在。对 DFMO 的不敏感是由于活性口袋中关键底物结合残基的取代。此外，在活性位点还鉴定到几个与抗酶抑制剂（AZI）相似的类似物，AZI 是 ODC 的非功能同源物。在这里，我们确立了 EhODC 序列保守了 PLP 结合残基的事实；相反，几个底物结合残基与 AZI 相似发生了突变。进一步的序列分析和结构研究表明，EhODC 可能代表活性脱羧酶和非活性 AZI 之间的进化桥梁。
