title: Cloning, expression and characterization of UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Wolbachia endosymbiont of human lymphatic filarial parasite Brugia malayi.
abstract: Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for treatment of lymphatic filariasis. Although functional characterization of the Wolbachia peptidoglycan assembly has not been fully explored, the Wolbachia genome provides evidence for coding all of the genes involved in lipid II biosynthesis, a part of peptidoglycan biosynthesis pathway. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is one of the lipid II biosynthesis pathway enzymes and it has inevitably been recognized as an antibiotic target. In view of the vital role of MurA in bacterial viability and survival, MurA ortholog from Wolbachia endosymbiont of Brugia malayi (wBm-MurA) was cloned, expressed and purified for further molecular characterization. The enzyme kinetics and inhibition studies were undertaken using fosfomycin. wBm-MurA was found to be expressed in all the major life stages of B. malayi and was immunolocalized in Wolbachia within the microfilariae and female adults by the confocal microscopy. Sequence analysis suggests that the amino acids crucial for enzymatic activity are conserved. The purified wBm-MurA was shown to possess the EPSP synthase (3-phosphoshikimate 1-carboxyvinyltransferase) like activity at a broad pH range with optimal activity at pH 7.5 and 37°C temperature. The apparent affinity constant (Km) for the substrate UDP-N-acetylglucosamine was found to be 0.03149 mM and for phosphoenolpyruvate 0.009198 mM. The relative enzymatic activity was inhibited ∼2 fold in presence of fosfomycin. Superimposition of the wBm-MurA homology model with the structural model of Haemophilus influenzae (Hi-MurA) suggests binding of fosfomycin at the same active site. The findings suggest wBm-MurA to be a putative antifilarial drug target for screening of novel compounds.
authors.affiliation: Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi, India; Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India.
authors.forename: Mohd
authors.initials: M
authors.lastname: Shahab
authors.affiliation: Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi, India; Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India.
authors.forename: Meenakshi
authors.initials: M
authors.lastname: Verma
authors.affiliation: Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India.
authors.forename: Manisha
authors.initials: M
authors.lastname: Pathak
authors.affiliation: Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi, India; Electron Microscopy, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India.
authors.forename: Kalyan
authors.initials: K
authors.lastname: Mitra
authors.affiliation: Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi, India; Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India.
authors.forename: Shailja
authors.initials: S
authors.lastname: Misra-Bhattacharya
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017178:Brugia malayi / Q000187:drug effects / Q000254:growth & development / Q000382:microbiology*; D003001:Cloning, Molecular; D005260:Female; D005368:Filariasis / Q000469:parasitology*; D005578:Fosfomycin / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008018:Life Cycle Stages; D008221:Lymphoid Tissue / Q000469:parasitology* / Q000473:pathology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D051189:Murinae; D010271:Parasites / Q000187:drug effects / Q000254:growth & development / Q000382:microbiology*; D010457:Peptidoglycan / Q000096:biosynthesis; D010802:Phylogeny; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D040681:Structural Homology, Protein; D013559:Symbiosis / Q000187:drug effects; D013696:Temperature; D020577:Wolbachia / Q000187:drug effects / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D010457:Peptidoglycan; D005578:Fosfomycin; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase