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. p stB 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-lauroy lsarcosine and was purified by gel permeation chromatography. The molecular mass of the protein was similar to 31 kDa. The eluted protein showed ATP-b inding ability and exhibited ATPase activity. Among different nucleotide tr iphosphates, ATP was found to be the preferred substrate for M. tuberculosi s PstB-ATPase. The study of the kinetics of ATP hydrolysis yielded K-m of s imilar to 72 muM and V-max of similar to0.12 mu mol/min/mg of protein. Diva lent cation like manganese was inhibitory to the ATPase activity. Magnesium or calcium, on the other hand, had no influence on the functionality of th e enzyme. The classical ATPase inhibitors like sodium azide, sodium vanadat e, and N-ethylmaleimide were without any effect but an ATP analogue, 5'-p-f luorosulfonylbenzoyl adenosine, inhibited the ATPase function of the recomb inant protein with a K-i of similar to0.40 mM. Furthermore, there was hardl y any ATP hydrolyzing ability of the PstB as a result of mutation of the co nserved aspartic acid residue to lysine in the Walker motif B, confirming t he recombinant protein is an ATPase. Interestingly, analysis of the recombi nant PstB revealed that it is a thermostable ATPase; thus, our results high light for the first time the presence of such an enzyme in any mesophilic b acteria.