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The glucomannokinase of Prevotella bryantii B₁4 and its potential role in regulating ß-glucanase expression

Department of Microbiology, Cornell University and ²Agricultural Research Service, USDA, Ithaca, NY 14853, USA

Author for correspondence: James B. Russell. Tel: +1 607 255 4508. Fax: +1 607 255 3904. e-mail: jbr8{at}cornell.edu

Prevotella bryantii B₁4 has a transport system for glucose and mannose, but ß-glucanase expression is only catabolite-repressed by glucose. P. bryantii B₁4 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 ß-glucanase activity increased. The glucose consumption rate and the glucomannokinase activity of cells treated with 2DG were highly correlated (r²=0·98). This result suggested that glucomannokinase activity was either directly or indirectly regulating ß-glucanase expression.

Keywords: rumen bacteria, sugar transport, regulation, catabolite repression

Abbreviations: 2-DG, 2-deoxyglucose; 2-ME, 2-mercaptoethanol; PEP, phosphoenolpyruvate

The SWISS-PROT accession number for the sequence of P. bryantii B₁4 glucomannokinase reported in this paper is P82680.

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