title: Trehalose-6-phosphate phosphorylase is part of a novel metabolic pathway for trehalose utilization in Lactococcus lactis.
abstract: Lactococcus lactis splits phosphorylated trehalose by the action of inorganic phosphate-dependent trehalose-6-phosphate phosphorylase (TrePP) in a novel catabolic pathway. TrePP was found to catalyze the reversible conversion of trehalose 6-phosphate into beta-glucose 1-phosphate and glucose 6-phosphate by measuring intermediate sugar phosphates in cell extracts from trehalose-cultivated lactococci. According to native PAGE and SDS-PAGE, TrePP was shown to be a monomeric enzyme with a molecular mass of 94 kDa. Reaction kinetics suggested that the enzyme follows a ternary complex mechanism with optimal phosphorolysis at 35 degrees C and pH 6.3. The equilibrium constants were found to be 0.026 and 0.032 at pH 6.3 and 7.0, respectively, favoring the formation of trehalose 6-phosphate. The Michaelis-Menten constants of TrePP for trehalose 6-phosphate, inorganic phosphate, beta-glucose 1-phosphate, and glucose 6-phosphate were determined to be 6, 32, 0.9, and 4 mm, respectively. The TrePP-encoding gene, designated trePP, was localized in a putative trehalose operon of L. lactis. This operon includes the gene encoding beta-phosphoglucomutase in addition to three open reading frames believed to encode a transcriptional regulator and two trehalose-specific phosphotransferase system components. The identity of trePP was confirmed by determining the N-terminal amino acid sequence of TrePP and by its overexpression in Escherichia coli and L. lactis, as well as the construction of a lactococcal trePP knockout mutant. Furthermore, both TrePP and beta-phosphoglucomutase activity were detected in Enterococcus faecalis cell extract, indicating that this bacterium exhibits the same trehalose assimilation route as L. lactis.
authors.affiliation: Applied Microbiology, Center for Chemistry and Chemical Engineering, Lund Institute of Technology, Lund University, P.O. Box 124, Lund SE-221 00, Sweden.
authors.forename: U
authors.initials: U
authors.lastname: Andersson
authors.forename: F
authors.initials: F
authors.lastname: Levander
authors.forename: P
authors.initials: P
authors.lastname: Rådström
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D015139:Blotting, Southern; D004591:Electrophoresis, Polyacrylamide Gel; D013293:Enterococcus faecalis / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000378:metabolism; D005958:Glucosephosphates / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000378:metabolism*; D008954:Models, Biological; D010733:Phosphoglucomutase / Q000737:chemistry / Q000235:genetics; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D013237:Stereoisomerism; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic; D014199:Trehalose / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D005958:Glucosephosphates; D019298:Glucose-6-Phosphate; D014199:Trehalose; C031590:glucose-1-phosphate; D005964:Glucosyltransferases; C442221:trehalose-6-phosphate phosphorylase; C023181:alpha,alpha-trehalose phosphorylase; D010733:Phosphoglucomutase