Cloning, expression, and characterization of polyphosphate glucokinase from Mycobacterium tuberculosis.
Polyphosphate glucokinase from Mycobacterium tuberculosis catalyzes the phosphorylation of glucose using polyphosphate or ATP as the phosphoryl donor. The M. tuberculosis H37Rv gene encoding this enzyme has been cloned, sequenced, and expressed in Escherichia coli. The gene contains an open reading frame for 265 amino acids with a calculated mass of 27,400 daltons. The recombinant polyphosphate glucokinase was purified 189-fold to homogeneity and shown to contain dual enzymatic activities, similar to the native enzyme from H37Ra strain. The high G+C content in the codon usage (64.5%) of the gene and the absence of an E. coli-like promoter consensus sequence are consistent with other mycobacterial genes. Two phosphate binding domains conserved in the eukaryotic hexokinase family were identified in the polyphosphate glucokinase sequence, however, "adenosine" and "glucose" binding motifs were not apparent. In addition, a putative polyphosphate binding region is also proposed for the polyphosphate glucokinase enzyme.
Hsieh
P C
PC
Department of Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4935, USA.
Shenoy
B C
BC
Samols
D
D
Phillips
N F
NF
D000241:Adenosine / Q000378:metabolism; D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D005941:Glucokinase / Q000737:chemistry; D005947:Glucose / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010770:Phosphotransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010770:Phosphotransferases; D005941:Glucokinase; C024812:polyphosphate-glucose phosphotransferase; D005947:Glucose; D000241:Adenosine