B-subunit of phosphate-specific transporter from Mycobacterium tuberculosis is a thermostable ATPase.
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.
Sarin
J
J
Institute of Microbial Technology, Sector 39A, Chandigarh 160 036, India.
Aggarwal
S
S
Chaba
R
R
Varshney
G C
GC
Chakraborti
P K
PK
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000502:physiology*; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D001426:Bacterial Proteins; D015139:Blotting, Southern; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002412:Cations; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008027:Light; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000378:metabolism; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002412:Cations; D011994:Recombinant Proteins; D008345:Manganese; C025231:sarkosyl; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C120060:phosphate import ATP-binding protein, Bacteria; D008274:Magnesium; D002118:Calcium; D012521:Sarcosine