Characterization of trehalose phosphorylase from Schizophyllum commune

Abstract

During growth on d-glucose, the basidiomycete Schizophyllum commune produces an intracellular alpha,alpha-trehalose phosphorylase. Specific phosphorylase activity increases steadily during the exponential growth phase, up to a maximum of approx. 0.08 unit/mg of protein, and decreases after the available d-glucose in the medium has been fully depleted. The variation with time of the concentrations of intracellular alpha,alpha-trehalose and Pi is reciprocal to that of trehalose phosphorylase activity, indicating that the enzyme makes temporary use of the pool of alpha, alpha-trehalose (approx. 0.42 mmol/g dry cell) via phosphorolysis. The enzyme has been purified, 150-fold, to homogeneity in 55% yield and characterized. It is a monomeric 61 kDa protein, which seems to lack regulation at the level of enzyme activity. The enzyme catalyses the reversible phosphorolysis of alpha,alpha-trehalose into alpha-d-glucose 1-phosphate and alpha-d-glucose in the absence of cofactors, with a catalytic-centre activity at 30 degrees C of 14 s(-1). Double-reciprocal analysis of the initial velocities for trehalose phosphorolysis and synthesis yields intersecting patterns, and no exchange reaction occurs between alpha-d-glucose 1-phosphate and the phosphate analogue arsenate. Therefore trehalose phosphorylase operates by a ternary-complex, rather than a Ping-Pong, kinetic mechanism. The specificity constants (kcat/Km) of phosphate (6000 M(-1).s(-1)) and alpha-d-glucose 1-phosphate (3500 M(-1).s(-1)) compared with those of alpha,alpha-trehalose (161 M(-1).s(-1)) and d-glucose (260 M(-1).s(-1)), together with the inhibition by NaCl, which is competitive with respect to phosphate with a Ki of 67 mM, suggest an important role for ionic enzyme-phosphate interactions in the catalytic mechanism of trehalose phosphorylase. The isolated enzyme requires alpha,alpha-trehalose (0.1-0.3 M) for its conformational stability.

Fungal trehalose phosphorylase: kinetic mechanism, pH-dependence of the reaction and some structural properties of the enzyme from Schizophyllum commune

“…The equilibrium constant for eqn (1) has a value of $ 0.15 at pH 6.6 and 30 mC [2], indicating that glucosyl transfer from phosphate to -glucose is favoured thermodynamically in itro. A role for trehalose phosphorylase in fungal physiology remains to be established.…”

“…A role for trehalose phosphorylase in fungal physiology remains to be established. Limited evidence available so far suggests a possible function of the enzyme in developmental processes [3], whereby the partial degradation of accumulated α,α-trehalose appears to be the physiological reaction [2]. Given that a role for α,α-trehalose as a storage carbohydrate has been questioned [4,5], a key function of trehalose phosphorylase in mainstream sugar catabolism appears unlikely.…”

“…Given that a role for α,α-trehalose as a storage carbohydrate has been questioned [4,5], a key function of trehalose phosphorylase in mainstream sugar catabolism appears unlikely. Trehalose phosphorylases from the basidiomycete fungi Agaricus bisporus [6], Grifola frondosa [7] and Schizophyllum commune [2] have been purified and characterized. They share a molecular mass of $ 60 kDa for the protein subunit, and occur as a monomer [2] or oligomers [6,7].…”

“…Trehalose phosphorylases from the basidiomycete fungi Agaricus bisporus [6], Grifola frondosa [7] and Schizophyllum commune [2] have been purified and characterized. They share a molecular mass of $ 60 kDa for the protein subunit, and occur as a monomer [2] or oligomers [6,7]. The primary structures of the enzymes from G. frondosa [8] and Pleurotus cajor-saju (TrEMBL accession no.…”

“…By analogy with retaining glycosidases, for which a large body of mechanistic and structural information is available (for a review, see [11]), α-retaining glucosyl transfer to and from phosphate is anticipated to occur through a double-displacement mechanism that involves two configurationally invertive steps (reviewed in [11,12]) : (1) cleavage of the carbon-oxygen bond of the glucosyl donor and formation of a covalent β-glucosyl-enzyme intermediate ; and (2) reaction of the intermediate with phosphate to yield Glc 1-P. Phospholysis of sucrose by sucrose phosphorylase is a well characterized example of the α-retaining phosphorylase mechanism [13], and the kinetics of this enzymic reaction are Ping Pong Bi Bi. Unlike sucrose phosphorylase, and unusually for a retaining GT, fungal trehalose phosphorylase follows a ternarycomplex kinetic mechanism in which both substrates must bind to the enzyme before a product is released [2]. Differences in the reaction mechanisms of sucrose phosphorylase and trehalose phosphorylase are probably reflected by the fact that sucrose phosphorylase has been classified into glycosyl hydrolase (GH) family 13 (clan GH-H) [14].…”