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, b ut beta -glucanase expression is only catabolite-repressed by glucose. P. b ryantii B(1)4 cell extracts had ATP-dependent gluco- and mannokinase activi ties, and significant phosphoenolpyruvate- or GTP-dependent hexose phosphor ylation was not observed. Mannose inhibited glucose phosphorylation (and vi ce versa), and activity gels indicated that a single protein was responsibl e 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 hex ose (mg protein)(-1) min(-1), respectively] and glucose was a better substr ate 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 g els indicated that it was a dimer (monomer 34.5 kDa). The N-terminus (25 re sidues) had an 8 amino acid segment that was homologous to other bacterial glucokinases. The glucomannokinase was competitively inhibited by the nonme tabolizable glucose analogue 2-deoxyglucose (2DG), and cells grown with glu cose and 2DG had lower rates of glucose consumption than did cells given on ly glucose. When the ratio of 2DG to glucose was increased, the glucose con sumption rate decreased and the beta -glucanase activity increased. The glu cose consumption rate and the glucomannokinase activity of cells treated wi th 2DG were highly correlated (r(2) = 0.98). This result suggested that glu comannokinase activity was either directly or indirectly regulating beta -g lucanase expression.