title: Molecular cloning, purification and characterization of Brugia malayi phosphoglycerate kinase.
abstract: Phosphoglycerate kinase (PGK) is a glycolytic enzyme present in many parasites. It has been reported as a candidate molecule for drug and vaccine developments. In the present study, a full-length cDNA encoding the Brugia malayi 3-phosphoglycerate kinase (BmPGK) with an open reading frame of 1.3 kb was isolated and PCR amplified and cloned. The exact size of the BmPGK's ORF is 1377 bps. The BmPGK gene was subcloned into pET-28a (+) expression vector, the expressed enzyme was purified by affinity column and characterized. The SDS-PAGE analysis revealed native molecular weight of recombinant Brugia malayi 3-phosphoglycerate kinase (rBmPGK) to be ∼45 kDa. The enzyme was found sensitive to temperature and pH, it showed maximum activity at 25 °C and pH 8.5. The Kmvalues for PGA and ATP were 1.77 and 0.967 mM, respectively. The PGK inhibitor, clorsulon and antifilarial drugs albendazole and ivermectin inhibited the enzyme. The specific inhibitor of PGK, clorsulon, competitively inhibited enzyme with Kivalue 1.88 μM. Albendazole also inhibited PGK competitively with Kivalue 35.39 μM. Further these inhibitory studies were confirmed by docking and molecular simulation of drugs with enzyme. Clorsulon interacted with substrate binding site with glutamine 37 as well as in hinge regions with aspartic acid 385 and valine 387 at ADP binding site. On the other hand albendazole interacted with asparagine 335 residues. These effects were in good association with binding interactions. Thus current study might help in designing and synthesis of effective inhibitors for this novel drug target and understanding their mode of interaction with the potent anthelmintic drugs.
authors.affiliation: Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi 221005, India.
authors.forename: Ranjeet
authors.initials: R
authors.lastname: Kumar
authors.affiliation: Division of Biochemistry, CSIR - Central Drug Research Institute, Lucknow 226001, India.
authors.forename: Pawan Kumar
authors.initials: PK
authors.lastname: Doharey
authors.affiliation: Division of Biochemistry, CSIR - Central Drug Research Institute, Lucknow 226001, India.
authors.forename: Jitendra Kumar
authors.initials: JK
authors.lastname: Saxena
authors.affiliation: Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi 221005, India. Electronic address: surathaur@rediffmail.com.
authors.forename: Sushma
authors.initials: S
authors.lastname: Rathaur
mesh_terms: D000818:Animals; D017178:Brugia malayi / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015801:Helminth Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016366:Open Reading Frames; D010735:Phosphoglycerate Kinase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D015801:Helminth Proteins; D011994:Recombinant Proteins; D010735:Phosphoglycerate Kinase
keywords: Brugia malayi; Clorsulon; Drug target; Lymphatic filariasis; Phosphoglycerate kinase