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J. Biol. Chem., Vol. 276, Issue 48, 44590-44597, November 30, 2001
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From the Institute of Microbial Technology, Sector 39A,
Chandigarh 160 036, India
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
~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
Km of ~72 µM and
Vmax of ~0.12 µmol/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 Ki of
~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.
B-subunit of Phosphate-specific Transporter from
Mycobacterium tuberculosis Is a Thermostable
ATPase*
,,
*
This work was supported in part by a research grant from the
Department of Biotechnology, New Delhi, India.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of a senior research fellowship from the Council of
Scientific and Industrial Research, New Delhi, India.
§
To whom correspondence should be addressed. Tel.: 91-172-695215 (ext. 452); Fax: 91-172-690585; E-mail:
pradip@imtech.res.in.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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