Abstract
Trehalose-6-phosphate phosphatase (TPP) is one of the primary enzymes involved in the synthesis of trehalose, the main sugar found in insect hemolymph. In the present study, we report for the first time heterologous expression in Pichia pastoris, characterization and homology modeling of TPP from Anopheles gambiae mosquito. Purified TPP recombinant exhibited a molecular weight of approximately 36 kDa with optimum pH of 8.0, optimum temperature of 38°C, and the KM was 3.19 ± 0.10 mM. Inhibition tests revealed that CaCl2 and Ca(NO3)2 at 5 and 25 mM, respectively, were effective inhibitors of TPP activity. Homology studies and molecular modeling indicated high similarity of the TPP tridimensional structure with TPP enzymes deposited at a data bank and these homology studies confirmed that it is a trehalose phosphatase with conserved motifs of the superfamily haloacid dehydrogenase. These results may provide support for the discovering of TPP inhibitors to be used for development of insecticides to control mosquito vectors. Key words: Trehalose, trehalose-6-phosphate phosphatase, expression, enzyme characterization, homology modeling.
Highlights
Malaria is a potentially serious infectious disease caused by unicellular protozoans of the genus Plasmodium species and is transmitted to vertebrate hosts through bites from females of the mosquito Anopheles species (Forattini, 2002; WHO, 2016)
Trehalose-6-phosphate phosphatase (TPP) is important for the metabolism and physiology of the mosquito A. gambiae, which is an important vector of Plasmodium falciparum, the parasite responsible for the most serious form of human malaria in Africa
TPP was chosen for cloning and expression because its catalytic product is trehalose, which is not essential for mammal cells, and not glucose, which is the major carbohydrate required for numerous mammalian metabolic pathways (Karthik et al, 2011; Klutts et al, 2003; Kormish and McGhee, 2005; Kushwaha et al, 2011)
Summary
Malaria is a potentially serious infectious disease caused by unicellular protozoans of the genus Plasmodium species and is transmitted to vertebrate hosts through bites from females of the mosquito Anopheles species (Forattini, 2002; WHO, 2016). Human malaria is a parasitic disease with medical, social and economic importance, and it constitutes one of the most important public health problems worldwide. Malaria is endemic in 91 countries, with 212 million estimated cases and 429 000 estimated deaths from malaria globally in 2015 (WHO, 2016). The vast majority of malaria deaths occur in Africa, especially among children (Garcia, 2010), and the mosquito Anopheles gambiae Giles, belonging to the subgenus Cellia, is the primary human malaria vector. Resistance in the mosquito vector to at least one of the insecticides used for malaria control has been detected in 60 countries (WHO, 2016). Studies on the metabolism of the insect vector and main enzymes involved in different metabolic pathways are important for collecting data on the insect's physiology, which can be used in the research and design of insect control strategies
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