Characterization and mutational analysis of an exopolyphosphatase from Mycobacterium smegmatis

Inorganic polyphosphate (poly-P) is a linear polymer comprising tens to hundreds of orthophosphate residues linked by ‘high-energy' phosphoanhydride bonds. These bio-polymers are found within all living cells, and have been shown to have a wide variety of important physiological functions. In most bacteria, the hydrolysis of Poly-P chains to monophosphate is predominantly mediated by exopolyphosphatase (Ppx) enzymes. However many bacterial species, lack identifiable Ppx enzymes. Mycobacterium smegmatis, an important model organism for tuberculosis-related experimental systems, lacks an identifiable Ppx protein, but encodes a protein sharing homology with both guanosine pentaphosphate hydrolase and Ppx enzymes (Msmeg_5413).

Objectives: To characterize the biochemical activities of the Msmeg_5413, and use a mutational approach to identify catalytically-important residues.

Methods: Sequence alignment analyzes were used to identify four highly-conserved residues in Msmeg_5413 (E121, D144, S149 and E151), putatively involved in poly-P hydrolysis. The corresponding alanine mutants were constructed. The wild-type and four mutant forms of Msmeg_5413 were cloned and expressed in E. coli; and the five purified recombinant proteins were subjected to comparative biochemical analysis.

Results: As predicted, Msmeg_5413 exhibited exopolyphsophatase activities. Notably, it also metabolized various nucleotide tri- and diphosphate substrates (NTPs/NDPs). All four putative active-site mutations markedly reduced, but did not abolish exopolyphosphatase activities; and variously affected NTP/NDP metabolic activities.

Conclusions: Results are consistent with Msmeg_5413 functioning as an exopolyphosphatase within M. smegmatis cells. Our mutational analysis provides significant mechanistic insight into this protein's biochemical activities, and putative physiological roles.

Funding: Research Grants Council of Hong Kong, GRF grant (#705007) to RMW