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 β-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 °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, β-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 β-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 inEscherichia coli and L. lactis, as well as the construction of a lactococcal trePP knockout mutant. Furthermore, both TrePP and β-phosphoglucomutase activity were detected in Enterococcus faecalis cell extract, indicating that this bacterium exhibits the same trehalose assimilation route asL. lactis.