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B-subunit of phosphate-specific transporter from Mycobacterium tuberculosis is a thermostable ATPase.

DIR@IMTECH: CSIR-Institute of Microbial Technology

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Title B-subunit of phosphate-specific transporter from Mycobacterium tuberculosis is a thermostable ATPase.
 
Creator Sarin, Jyoti
Aggarwal, S
Chaba, Rachna
Varshney, Grish C
Chakraborti, Pradip K
 
Subject QD Chemistry
 
Description The B-subunit of phosphate-specific transporter (PstB) is an ABC protein. pstB was polymerase chain reaction-amplified from Mycobacterium tuberculosis and overexpressed in Escherichia coli. The overexpressed protein was found to be in inclusion bodies. The protein was solubilized using 1.5% N-lauroylsarcosine and was purified by gel permeation chromatography. The molecular mass of the protein was approximately 31 kDa. The eluted protein showed ATP-binding ability and exhibited ATPase activity. Among different nucleotide triphosphates, ATP was found to be the preferred substrate for M. tuberculosis PstB-ATPase. The study of the kinetics of ATP hydrolysis yielded K(m) of approximately 72 microm and V(max) of approximately 0.12 micromol/min/mg of protein. Divalent cation like manganese was inhibitory to the ATPase activity. Magnesium or calcium, on the other hand, had no influence on the functionality of the enzyme. The classical ATPase inhibitors like sodium azide, sodium vanadate, and N-ethylmaleimide were without any effect but an ATP analogue, 5'-p-fluorosulfonylbenzoyl adenosine, inhibited the ATPase function of the recombinant protein with a K(i) of approximately 0.40 mm. Furthermore, there was hardly any ATP hydrolyzing ability of the PstB as a result of mutation of the conserved aspartic acid residue to lysine in the Walker motif B, confirming the recombinant protein is an ATPase. Interestingly, analysis of the recombinant PstB revealed that it is a thermostable ATPase; thus, our results highlight for the first time the presence of such an enzyme in any mesophilic bacteria.
 
Publisher ASBMB
 
Date 2001-11-30
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://crdd.osdd.net/open/288/1/chakraborti2001.pdf
Sarin, Jyoti and Aggarwal, S and Chaba, Rachna and Varshney, Grish C and Chakraborti, Pradip K (2001) B-subunit of phosphate-specific transporter from Mycobacterium tuberculosis is a thermostable ATPase. The Journal of biological chemistry, 276 (48). pp. 44590-7. ISSN 0021-9258
 
Relation http://www.jbc.org/content/276/48/44590.full.pdf+html
http://crdd.osdd.net/open/288/