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Title of Journal: Curr Microbiol

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Abbravation: Current Microbiology

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Springer US

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DOI

10.1002/pssb.2220580244

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1432-0991

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Molecular Characterization of Oxidative StressInd

Authors: Gurdyal Singh Stuti Arya Arbind Kumar Dominic Narang Jagdeep Kaur
Publish Date: 2013/11/14
Volume: 68, Issue: 3, Pages: 387-396
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Abstract

The Mycobacterium tuberculosis has developed intricate strategies to evade the killing of microorganism and support its survival in phagocytes The genome sequence of bacterium revealed the presence of several genes for lypolytic enzymes Rv1923 gene a member of Lip family in M tuberculosis demonstrated the least sequence similarity with its counterpart in nonpathogenic strain M smegmatis The expression of Rv1923 gene LipD was not observed in in vitro growing cultures of M tuberculosis H37Ra while an upregulation of transcription of Rv1923 gene was noticed in oxidative conditions For detailed characterization of LipD enzyme the Rv1923 gene was cloned in pQE30UA vector and expressed in E coli M15 cells LipD was purified from inclusion bodies and refolded with nearly 40  protein yield The specific activity of enzyme was calculated to be 16 U/mg with pNPpalmitate as a preferred substrate Kinetic analysis showed K m 0645 mM and V max 2475 U/ml with pNPpalmitate Ser102 Asp342 and His369 predicted as the members of the catalytic triad were confirmed by mutagenesis Mutagenesis studies revealed that catalytic serine residues located in βlactamase motifs SXXK were responsible for lipolytic activity Secondary structure analysis by CD spectroscopy demonstrated the presence of α helices and β sheets in the canonical structure of LipD The enzyme was stable up to 50 °C and was active even at pH 60 The expression of enzyme under stress conditions and its activity and stability at high temperature and low pH suggested the possible role of LipD in the survival of mycobacterium in macrophage compartmentFinancial support from Department of Biotechnology New Delhi and Council of Scientific and Industrial Research New Delhi India to JK and SRF to GS is acknowledged We thank Dr S Mukhopadhyay from IISER SAS Nagar Mohali for providing the facility for spectroscopic analysis

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