A glucose kinase from Mycobacterium smegmatis.
Carbon metabolism and regulation is poorly understood in mycobacteria, a genus that includes some major pathogenic species like Mycobacterium tuberculosis and Mycobacterium leprae. Here, we report the identification of a glucose kinase from Mycobacterium smegmatis. This enzyme serves in glucose metabolism and global carbon catabolite repression in the related actinomycete Streptomyces coelicolor. The gene, msmeg1356 (glkA), was found by means of in silico screening. It was shown that it occurs in the same genetic context in all so far sequenced mycobacterial species, where it is located in a putative tricistronic operon together with a glycosyl hydrolase and a putative malonyl-CoA transacylase. Heterologous expression of glkA in an Escherichia coli glucose kinase mutant led to the restoration of glucose growth, which provided in vivo evidence for glucose kinase function. GlkA(Msm) was subsequently overproduced in order to study its enzymatic features. We found that it can form a dimer and that it efficiently phosphorylates glucose at the expense of ATP. The affinity constant for glucose was with 9 mM about eight times higher and the velocity was about tenfold slower when compared to the parallel measured glucose kinase of S. coelicolor. Both enzymes showed similar substrate specificity, which consists in an ATP-dependent phosphorylation of glucose and no, or very inefficient, phosphorylation of the glucose analogues 2-deoxyglucose and methyl alpha-glucoside. Hence, our data provide a basis for studying the role of mycobacterial glucose kinase in vivo to unravel possible catalytic and regulatory functions.
Pimentel-Schmitt
Elisângela F
EF
Department of Microbiology, Friedrich Alexander University Erlangen-Nurnberg, Erlangen, Germany.
Thomae
Andreas W
AW
Amon
Johannes
J
Klieber
Michael A
MA
Roth
Heide-Marie
HM
Muller
Yves A
YA
Jahreis
Knut
K
Burkovski
Andreas
A
Titgemeyer
Fritz
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D005941:Glucokinase / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics; D009876:Operon; D010766:Phosphorylation; D010802:Phylogeny; D013379:Substrate Specificity
D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; C096446:GlkA protein, Bacteria; D005941:Glucokinase; D005947:Glucose