Glucose kinase of Streptomyces coelicolor A3(2): large-scale purification and biochemical analysis.
Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.
Mahr
K
K
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
van Wezel
G P
GP
Svensson
C
C
Krengel
U
U
Bibb
M J
MJ
Titgemeyer
F
F
D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D005941:Glucokinase