Ebrecht Ana C, Asención Diez Matías D, Piattoni Claudia V, Guerrero Sergio A, Iglesias Alberto A
Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Ciudad Universitaria, Paraje "El Pozo" CC 242, S3000ZAA Santa Fe, Argentina.
Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Ciudad Universitaria, Paraje "El Pozo" CC 242, S3000ZAA Santa Fe, Argentina.
Biochim Biophys Acta. 2015 Jan;1850(1):88-96. doi: 10.1016/j.bbagen.2014.10.002. Epub 2014 Oct 12.
Giardia lamblia is a pathogen of humans and other vertebrates. The synthesis of glycogen and of structural oligo and polysaccharides critically determine the parasite's capacity for survival and pathogenicity. These characteristics establish that UDP-glucose is a relevant metabolite, as it is a main substrate to initiate varied carbohydrate metabolic routes.
Herein, we report the molecular cloning of the gene encoding UDP-glucose pyrophosphorylase from genomic DNA of G. lamblia, followed by its heterologous expression in Escherichia coli. The purified recombinant enzyme was characterized to have a monomeric structure. Glucose-1-phosphate and UTP were preferred substrates, but the enzyme also used galactose-1-phosphate and TTP. The catalytic efficiency to synthesize UDP-galactose was significant. Oxidation by physiological compounds (hydrogen peroxide and nitric oxide) inactivated the enzyme and the process was reverted after reduction by cysteine and thioredoxin. UDP-N-acetyl-glucosamine pyrophosphorylase, the other UTP-related enzyme in the parasite, neither used galactose-1-phosphate nor was affected by redox modification.
Our results suggest that in G. lamblia the UDP-glucose pyrophosphorylase is regulated by oxido-reduction mechanism. The enzyme exhibits the ability to synthesize UDP-glucose and UDP-galactose and it plays a key role providing substrates to glycosyl transferases that produce oligo and polysaccharides.
The characterization of the G. lamblia UDP-glucose pyrophosphorylase reinforces the view that in protozoa this enzyme is regulated by a redox mechanism. As well, we propose a new pathway for UDP-galactose production mediated by the promiscuous UDP-glucose pyrophosphorylase of this organism.
蓝氏贾第鞭毛虫是人类和其他脊椎动物的病原体。糖原以及结构性寡糖和多糖的合成对于该寄生虫的生存和致病能力至关重要。这些特性表明UDP-葡萄糖是一种重要的代谢物，因为它是启动各种碳水化合物代谢途径的主要底物。
在此，我们报道了从蓝氏贾第鞭毛虫基因组DNA中克隆编码UDP-葡萄糖焦磷酸化酶的基因，并在大肠杆菌中进行了异源表达。纯化的重组酶具有单体结构。葡萄糖-1-磷酸和UTP是其优选底物，但该酶也可利用半乳糖-1-磷酸和TTP。合成UDP-半乳糖的催化效率显著。生理化合物（过氧化氢和一氧化氮）的氧化使该酶失活，而在半胱氨酸和硫氧还蛋白还原后该过程得以逆转。寄生虫中的另一种与UTP相关的酶UDP-N-乙酰葡糖胺焦磷酸化酶既不利用半乳糖-1-磷酸，也不受氧化还原修饰的影响。
我们的结果表明，在蓝氏贾第鞭毛虫中，UDP-葡萄糖焦磷酸化酶受氧化还原机制调控。该酶具有合成UDP-葡萄糖和UDP-半乳糖的能力，并且在为产生寡糖和多糖的糖基转移酶提供底物方面发挥关键作用。
蓝氏贾第鞭毛虫UDP-葡萄糖焦磷酸化酶的特性强化了原生动物中该酶受氧化还原机制调控的观点。此外，我们提出了由该生物体中混杂的UDP-葡萄糖焦磷酸化酶介导的UDP-半乳糖产生的新途径。
