The glucomannokinase of Prevotella bryantii B(1)4 and its potential role in regulating beta-glucanase expression.
Prevotella bryantii B(1)4 has a transport system for glucose and mannose, but beta-glucanase expression is only catabolite-repressed by glucose. P bryantii B(1)4 cell extracts had ATP-dependent gluco- and mannokinase activities, and significant phosphoenolpyruvate- or GTP-dependent hexose phosphorylation was not observed. Mannose inhibited glucose phosphorylation (and vice versa), and activity gels indicated that a single protein was responsible for both activities. Glucose was phosphorylated at a faster rate than was mannose [V(max) 280 nmol hexose (mg protein)(-1) min(-1) versus 60 nmol hexose (mg protein)(-1) min(-1), respectively] and glucose was a better substrate for the kinase (K(m) 0.12 mM versus 1.2 mM, respectively). The purified glucomannokinase (1250-fold) had a molecular mass of 68 kDa, but SDS-PAGE gels indicated that it was a dimer (monomer 34.5 kDa). The N-terminus (25 residues) had an 8 amino acid segment that was homologous to other bacterial glucokinases. The glucomannokinase was competitively inhibited by the nonmetabolizable glucose analogue 2-deoxyglucose (2DG), and cells grown with glucose and 2DG had lower rates of glucose consumption than did cells given only glucose. When the ratio of 2DG to glucose was increased, the glucose consumption rate decreased and the beta-glucanase activity increased. The glucose consumption rate and the glucomannokinase activity of cells treated with 2DG were highly correlated (r(2)=0.98). This result suggested that glucomannokinase activity was either directly or indirectly regulating beta-glucanase expression.
Fields
Matthew W
MW
Department of Microbiology, Cornell University and 2Agricultural Research Service, USDA, Ithaca, NY 14853, USA.
Russell
James B
JB
Department of Microbiology, Cornell University and 2Agricultural Research Service, USDA, Ithaca, NY 14853, USA.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002480:Cellulase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D005941:Glucokinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018720:Prevotella / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D005941:Glucokinase; C032922:mannokinase; D002480:Cellulase; D005947:Glucose; D008358:Mannose