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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. GlkAMsm 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 α-glucoside. Hence, our data provide a basis for studying the role of mycobacterial glucose kinase in vivo to unravel possible catalytic and regulatory functions. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Members of the soil-dwelling prokaryotic genus Streptomyces are indispensable for the recycling of complex polysaccharides, and produce a wide range of natural products. Nutrient limitation is likely to be a major signal for the onset of their development, resulting in spore formation by specialized aerial hyphae. Streptomycetes grow on numerous carbon sources, which they utilize in a preferential manner. The main signaling pathway underlying this phenomenon is carbon catabolite repression, which in streptomycetes is totally dependent on the glycolytic enzyme glucose kinase (Glk). How Glk exerts this fascinating dual role (metabolic and regulatory) is still largely a mystery. We show here that while Glk is made constitutively throughout the growth of Streptomyces coelicolor A3(2), its catalytic activity is modulated in a carbon source-dependent manner: while cultures growing exponentially on glucose exhibit high Glk activity, mannitol- grown cultures show negligible activity. Glk activity was directly proportional to the amount of two Glk isoforms observed by Western blot analysis. The activity profile of GlcP, the major glucose permease, correlated very well with that of Glk. Our data are consistent with a direct interaction between Glk and GlcP, suggesting that a Glk-GlcP permease complex is required for efficient glucose transport by metabolic trapping. This is supported by the strongly reduced accumulation of glucose in glucose kinase mutants. A model to explain our data is presented. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]