Isolation, characterization, interaction of a thiazolekinase (Plasmodium falciparum) with silver nanoparticles

• Published in: International journal of biological macromolecules Vol. 79; pp. 644 - 653
• Main Authors: Yao, J., van Marwijk, J., Wilhelmi, B., Whiteley, C.G.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-08-2015
• Subjects: 5-(2-Hydroxyethyl)-4-methyl-thioazolekinase; Amino Acid Sequence; Antimalarials - chemistry; Antimalarials - pharmacology; Binding Sites; Chromatography, Gel; Cloning, Molecular; Enzyme Assays; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Humans; Inhibition; Kinetics; Metal Nanoparticles - chemistry; Models, Molecular; Molecular Sequence Data; Molecular Weight; Phosphotransferases (Alcohol Group Acceptor) - antagonists & inhibitors; Phosphotransferases (Alcohol Group Acceptor) - chemistry; Phosphotransferases (Alcohol Group Acceptor) - genetics; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Plasmodium falciparum - drug effects; Plasmodium falciparum - enzymology; Plasmodium falciparum - genetics; Protein Binding; Protein Interaction Domains and Motifs; Protein Subunits - antagonists & inhibitors; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Protozoan Proteins - antagonists & inhibitors; Protozoan Proteins - chemistry; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Sequence Alignment; Silver - chemistry; Silver - pharmacology; Silver nanoparticles; Species Specificity; Thiamine - antagonists & inhibitors; Thiamine - biosynthesis; Silver nanoparticles; 5-(2-Hydroxyethyl)-4-methyl-thioazolekinase; Inhibition
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2015.05.033

•Recombinant His-tagged thiazole kinase isolated, purified from genomic DNA of Plasmodium falciparum.•Monomeric enzyme with mass 34kDa, specific activity 295nmolmg−1 temperature and pH optima of 37°C and 7.5.•Km (65μM) and Vmax (36.8μmolml−1min−1) purified by FPLC on Sephacryl S100HR.•Non-competitive inhibition by Ag (79%; Ki=6.45μM).•Nanoparticles interact through Met1, Cys206 on surface of enzyme.
Malaria, a mosquito-borne infectious disease, is caused by the Plasmodium genus, and remains one of the greatest health challenges worldwide. The malarial parasite possess a biosynthetic pathway for the B-group vitamin incorporating the thiamine metabolizing enzymes; humans on the other hand cannot synthesize the vitamin and require it from within their diet. The vitamin B1 biosynthetic enzyme 5-(2-hydroxyethyl)-4-methylthioazolekinase [EC. 2.7.1.50] from Plasmodium (PfThzK) is particularly attractive as a biomedical target since any inhibition of this enzyme may lead to an effective treatment for malaria. In the present study, PfThzK was recombinantly produced as a 6× His fusion protein in Escherichia coli BL21(DE3) and purified using nickel affinity and size exclusion chromatography. The enzyme was monomeric with a molecular mass of 34kDa, a specific activity of 295.04nmolmin−1mg−1 and showed an optimum temperature and pH of 37°C and 7.5, respectively. The purified PfThzK was non-competitively inhibited (79%) by silver nanoparticles (2–6nm); Ki=6.45μM. A mechanism is suggested for the interaction of the silver nanoparticle with PfThzK through two sulphur bearing amino acids (Met1, Cys206) on the surface of each subunit of the enzyme.