B-subunit of Phosphate-specific Transporter fromMycobacterium tuberculosis Is a Thermostable ATPase

Jyoti Sarin,Sita Aggarwal,Rachna Chaba,Grish C Varshney,Pradip K Chakraborti

doi:10.1074/jbc.m105401200

Jyoti Sarin, Sita Aggarwal + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m105401200

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Abstract

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.

Highlights

Importance of phosphate as an essential component of several biomolecules, such as membrane lipids, complex carbohydrates, nucleic acids, etc., is well known
Bacterial ATP-binding cassette (ABC) proteins have been shown to be responsible for ATP binding as well as ATP hydrolysis, which is evident from the studies with histidine and maltose transporters from S. typhimurium [16, 20, 21]
Phosphate-specific transporter (Pst) system is one of such importers belonging to the superfamily of ABC permeases and is known to be operative in bacteria in phosphate-limiting conditions [5]

Summary

The abbreviations used are

Phosphate-specific transporter; ABC, ATP-binding cassette; ATPase, adenosine triphosphatase; FSBA, 5Ј-p-fluorosulfonylbenzoyl adenosine; IgG, immunoglobulin G; IPTG, isopropyl-␤-D-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Pi, inorganic phosphate; PstB, B subunit of phosphate-specific transporter. PstB subunit, which is often referred as ABC protein [16], provides energy for transport through ATP hydrolysis [5]. Available reports indicated the similar organization of the genes of the pst operon in other prokaryotes [17, 18] as well, except for mycobacteria [7, 19]. Bacterial ABC proteins have been shown to be responsible for ATP binding as well as ATP hydrolysis, which is evident from the studies with histidine and maltose transporters from S. typhimurium [16, 20, 21]. We report that, unlike other prokaryotic ABC proteins, the ATP hydrolyzing ability of PstB from M. tuberculosis is rather magnesium-independent and resistant to known ATPase inhibitors. Our results convincingly established that the mycobacterial PstB is a thermostable ATPase and highlighted the presence of such an enzyme in any mesophilic bacteria

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION