title: Functional analysis of TPM domain containing Rv2345 of Mycobacterium tuberculosis identifies its phosphatase activity.
abstract: Mycobacterium tuberculosis (Mtb) is the causal agent of tuberculosis, the second largest infectious disease. With the rise of multi-drug resistant strains of M. tuberculosis, serious challenge lies ahead of us in treating the disease. The availability of complete genome sequence of Mtb has improved the scope for identifying new proteins that would not only further our understanding of biology of the organism but could also serve to discover new drug targets. In this study, Rv2345, a hypothetical membrane protein of M. tuberculosis H37Rv, which is reported to be a putative ortholog of ZipA cell division protein has been assigned function through functional annotation using bioinformatics tools followed by experimental validation. Sequence analysis showed Rv2345 to have a TPM domain at its N-terminal region and predicted it to have phosphatase activity. The TPM domain containing region of Rv2345 was cloned and expressed using pET28a vector in Escherichia coli and purified by Nickel affinity chromatography. The purified TPM domain was tested in vitro and our results confirmed it to have phosphatase activity. The enzyme activity was first checked and optimized with pNPP as substrate, followed by using ATP, which was also found to be used as substrate by the purified protein. Hence sequence analysis followed by in vitro studies characterizes TPM domain of Rv2345 to contain phosphatase activity.
authors.affiliation: Department of Biomedical Science, Acharya Narendra Dev College, University of Delhi, New Delhi 110019, India.
authors.forename: Avni
authors.initials: A
authors.lastname: Sinha
authors.affiliation: Department of Biomedical Science, Acharya Narendra Dev College, University of Delhi, New Delhi 110019, India.
authors.forename: Kandasamy
authors.initials: K
authors.lastname: Eniyan
authors.affiliation: School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Swati
authors.initials: S
authors.lastname: Sinha
authors.affiliation: School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Andrew Michael
authors.initials: AM
authors.lastname: Lynn
authors.affiliation: Department of Biomedical Science, Acharya Narendra Dev College, University of Delhi, New Delhi 110019, India. Electronic address: urmibajpai@andc.du.ac.in.
authors.forename: Urmi
authors.initials: U
authors.lastname: Bajpai
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D010744:Phosphoric Monoester Hydrolases
keywords: Cell division; Mycobacterium tuberculosis; Phosphatase; TPM domain