CLONING, EXPRESSION, AND CHARACTERIZATION OF POLYPHOSPHATE GLUCOKINASE FROM MYCOBACTERIUM-TUBERCULOSIS

Polyphosphate glucokinase from Mycobacterium tuberculosis catalyzes th e phosphorylation of glucose using polyphosphate or ATP as the phospho ryl donor. The M. tuberculosis H(37)Rv gene encoding this enzyme has b een cloned, sequenced, and expressed in Escherichia coli. The gene con tains an open reading frame for 265 amino acids with a calculated mass of 27,400 daltons. The recombinant polyphosphate glucokinase was puri fied 189-fold to homogeneity and shown to contain dual enzymatic activ ities, similar to the native enzyme from H37Ra strain. The high G+C co ntent in the codon usage (64.5%) of the gene and the absence of an E. coli-like promoter consensus sequence are consistent with other mycoba cterial genes. Two phosphate binding domains conserved in the eukaryot ic hexokinase family were identified in the polyphosphate glucokinase sequence, however, ''adenosine'' and ''glucose'' binding motifs were n ot apparent. In addition, a putative polyphosphate binding region is a lso proposed for the polyphosphate glucokinase enzyme.