Forero-Baena Nicolás, Sánchez-Lancheros Diana, Buitrago July Constanza, Bustos Victor, Ramírez-Hernández María Helena
Department of Chemistry, Universidad Nacional de Colombia, Bogotá Cundinamarca, Colombia.
Biochim Open. 2015 Nov 18;1:61-69. doi: 10.1016/j.biopen.2015.11.001. eCollection 2015.
is an intestinal protozoan parasite that causes giardiasis, a disease of high prevalence in Latin America, Asia and Africa. Giardiasis leads to poor absorption of nutrients, severe electrolyte loss and growth retardation. In addition to its clinical importance, this parasite is of special biological interest due to its basal evolutionary position and simplified metabolism, which has not been studied thoroughly. One of the most important and conserved metabolic pathways is the biosynthesis of nicotinamide adenine dinucleotide (NAD). This molecule is widely known as a coenzyme in multiple redox reactions and as a substrate in cellular processes such as synthesis of Ca mobilizing agents, DNA repair and gene expression regulation. There are two pathways for NAD biosynthesis, which converge at the step catalyzed by nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18). Using bioinformatics tools, we found two NMNAT sequences in ( and ). We first verified the identity of the sequences . Subsequently, was cloned into an expression vector. The recombinant protein (His-GlNMNAT) was purified by nickel-affinity binding and was used in direct enzyme assays assessed by C18-HPLC, verifying adenylyltransferase activity with both nicotinamide (NMN) and nicotinic acid (NAMN) mononucleotides. Optimal reaction pH and temperature were 7.3 and 26 °C. Michaelis-Menten kinetics were observed for NMN and ATP, but saturation was not accomplished with NAMN, implying low affinity yet detectable activity with this substrate. Double-reciprocal plots showed no cooperativity for this enzyme. This represents an advance in the study of NAD metabolism in spp.
是一种肠道原生动物寄生虫，可引起贾第虫病，该病在拉丁美洲、亚洲和非洲高发。贾第虫病会导致营养吸收不良、严重电解质流失和生长发育迟缓。除了其临床重要性外，由于其基础进化地位和简化的代谢方式，这种寄生虫还具有特殊的生物学研究价值，但尚未得到充分研究。烟酰胺腺嘌呤二核苷酸（NAD）的生物合成是最重要且保守的代谢途径之一。该分子作为多种氧化还原反应中的辅酶以及细胞过程（如钙动员剂合成、DNA修复和基因表达调控）中的底物广为人知。NAD生物合成有两条途径，它们在由烟酰胺/烟酸单核苷酸腺苷酸转移酶（NMNAT，EC 2.7.7.1/18）催化的步骤处汇合。使用生物信息学工具，我们在（和）中发现了两个NMNAT序列。我们首先验证了这些序列的身份。随后，将其克隆到表达载体中。重组蛋白（His-GlNMNAT）通过镍亲和结合进行纯化，并用于通过C18-HPLC评估的直接酶活性测定，验证了其对烟酰胺（NMN）和烟酸（NAMN）单核苷酸的腺苷酸转移酶活性。最佳反应pH和温度分别为7.3和26℃。观察到NMN和ATP符合米氏动力学，但NAMN未达到饱和，这意味着该底物亲和力低但仍可检测到活性。双倒数图显示该酶无协同性。这代表了对贾第虫属物种NAD代谢研究的一项进展。
