Substrate-binding recognition and specificity of trehalose phosphorylase from Schizophyllum commune examined in steady-state kinetic studies with deoxy and deoxyfluoro substrate analogues and inhibitors

Abstract

Trehalose phosphorylase is a component of the α--glucopyranosyl α--glucopyranoside (α,α-trehalose)-degrading enzyme system in fungi and it catalyses glucosyl transfer from α,α-trehalose to phosphate with net retention of the anomeric configuration. The enzyme active site has no detectable affinity for α,α-trehalose in the absence of bound phosphate and catalysis occurs from the ternary complex. To examine the role of non-covalent enzyme-substrate interactions for trehalose phosphorylase recognition, we used the purified enzyme from Schizophyllum commune and tested a series of incompetent structural analogues of the natural substrates and products as inhibitors of the enzyme. Equilibrium-binding constants (K i ) for deoxy-and deoxyfluoro derivatives of -glucose show that loss of interactions with the 3-, 4-or 6-OH, but not the reactive 1-and the 2-OH, results in considerably ( 100-fold) weaker affinity for sugar-binding subsite j1, revealing the requirement for hydrogen bonding with hydroxyls, away from the site of chemical transformation to position precisely the -glucose-leaving group\nucleophile for catalysis. The high specificity of trehalose phosphorylase for the sugar aglycon during binding and conversion of O-glycosides is in contrast with the observed α-retaining

Engineering an Antibiotic to Fight Cancer: Optimization of the Novobiocin Scaffold to Produce Anti-proliferative Agents

“…Numerous biologically active natural products contain carbohydrates appended to their scaffolds that serve to increase solubility and provide interactions with their cognate receptor. In many cases, removal of the carbohydrate moiety renders the aglycon inactive, whereas alteration of the sugar ring size can alter affinity of the compounds toward specific targets. − Modifications to the noviose pyranose ring were proposed to elucidate functionalities required for inhibitory activity as well as to determine whether different sized sugars can be utilized as replacements for noviose. Thus, five-, six-, and seven-membered sugars were synthesized and coupled to the aforementioned scaffolds to determine optimal interactions.…”

Structure–function relationships for Schizophyllum commune trehalose phosphorylase and their implications for the catalytic mechanism of family GT-4 glycosyltransferases

“…The available biochemical evidence for the wild-type enzyme [10,[14][15][16] strongly promotes studies of the ScTPase catalytic mechanism with site-directed mutagenesis. To provide the required structural basis, we report here on the isolation of the cDNA encoding ScTPase and the efficient heterologous expression thereof in Escherichia coli, as well as the detailed characterization of the purified recombinant enzyme.…”

“…Strong inhibition of amylolytic enzymes by the pseudo-tetrasaccharide acarbose requires that the acarviosine moiety, as shown in Figure 3, is intact [40]. There is overlap in binding recognition of subsites − 1 and + 1 in ScTPase [15,16] and therefore the orientation of validoxylamine A in the active centre is not unambiguously defined. The conformation of the hydroxymethylconduritol unit of the inhibitor is constrained by the double bond between C-5 and C-7 (which corresponds to the endocyclic O-5 in glucose) to a 2 H 3 half-chair in which atoms C-4, C-5, C-7 and C-1 are co-planar ( Figure 3).…”

Phosphate Analogues as Probes of the Catalytic Mechanisms of MurA and AroA, Two Carboxyvinyl Transferases

“…To probe the role of phosphate in catalysis, we studied the reverse reactions of AroA and MurA in the presence of phosphate analogues. Phosphate analogues have often been used as mechanistic probes for both P-O bond-cleaving enzymes (47)(48)(49)(50)(51)(52)(53)(54)(55) and C-O bond-cleaving enzymes (56)(57)(58)(59)(60)(61)(62).…”