The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies

Abstract

Glycosyltransferases (GTs) constitute a diverse class of enzymes that catalyze the synthesis of glycosidic bonds in oligosaccharides and glycoconjugates. A nucleotide-, phospho-or lipid-phospho-activated sugar is typically utilized as the donor substrate, and transfer of the glycosyl moiety to the acceptor molecule occurs with either inversion or retention of configuration at the reactive anomeric carbon [1]. After detailed studies of glycogen phosphorylase spanning many decades [2-6], there has been recent rekindled interest in the mechanistic characterization of retaining glycosyltransferases, particularly in relation to glycoside hydrolases, the physiological counterpart enzymes that catalyze the breakdown of glycosidic linkages [7]. The canonicalSchizophyllum commune a,a-trehalose phosphorylase utilizes a glycosyltransferase-like catalytic mechanism to convert its disaccharide substrate into a-d-glucose 1-phosphate and a-d-glucose. Recruitment of phosphate by the free enzyme induces a,a-trehalose binding recognition and promotes the catalytic steps. Like the structurally related glycogen phosphorylase and other retaining glycosyltransferases of fold family GT-B, the trehalose phosphorylase contains an Arg507-XXXX-Lys512 consensus motif (where X is any amino acid) comprising key residues of its putative phosphatebinding sub-site. Loss of wild-type catalytic efficiency for reaction with phosphate (k cat ⁄ K m = 21 000 m )1 AEs )1 ) was dramatic ( ‡10 7 -fold) in purified Arg507 fi Ala (R507A) and Lys512 fi Ala (K512A) enzymes, reflecting a corresponding change of comparable magnitude in k cat (Arg507) and K m (Lys512). External amine and guanidine derivatives selectively enhanced the activity of the K512A mutant and the R507A mutant respectively. Analysis of the pH dependence of chemical rescue of the K512A mutant by propargylamine suggested that unprotonated amine in combination with H 2 PO 4 ) , the protonic form of phosphate presumably utilized in enzymatic catalysis, caused restoration of activity. Transition state-like inhibition of the wild-type enzyme A by vanadate in combination with a,a-trehalose (K i = 0.4 lm) was completely disrupted in the R507A mutant but only weakened in the K512A mutant (K i = 300 lm). Phosphate (50 mm) enhanced the basal hydrolase activity of the K512A mutant toward a,a-trehalose by 60% but caused its total suppression in wild-type and R507A enzymes. The results portray differential roles for the side chains of Lys512 and Arg507 in trehalose phosphorylase catalysis, reactant state binding of phosphate and selective stabilization of the transition state respectively.Abbreviations G1P, a-D-glucose 1-phosphate; GTs, glycosyltransferases; K512A, Lys512 fi Ala mutant; R507A, Arg507 fi Ala mutant; ScTPase, Schizophyllum commune trehalose phosphorylase; S N i-like, internal return-like mechanism.

Carbohydrate synthesis by disaccharide phosphorylases: Reactions, catalytic mechanisms and application in the glycosciences

“…3). Mutagenesis data confirm the importance of Lys-512 and Arg-507 as key phosphate-binding residues for the activity of trehalose phosphorylase [32]. Both trehalose and glycogen phosphorylase lack a suitable amino acid in their respective active site whose side chain would be positioned appropriately to be a candidate catalytic nucleophile.…”

Structural and Functional Analysis of a New Subfamily of Glycosyltransferases Required for Glycosylation of Serine-rich Streptococcal Adhesins

“…The side chain density of Arg-179 is high in molecules B and D but is very low in molecules A and C in the Gtf3 tetramer (no density for C␤ to C␦ and not well defined NH1 and NH 2 densities), likely a result of movement during catalysis, which may have occurred in the substrate-cocrystallized crystals. This type of interaction between an arginine side chain and the ␤-phosphate atoms is considered to be a conserved feature of the retaining glycosyltransferase (33,36,37). The arginine side chain presumably neutralizes the negative charge that develops on the ␤-phosphate during catalysis.…”

Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

“…The dissociative nature of the transition state may be further stabilized by Arg-261, which forms an ion pair with the ␤-phosphate. A comparison of the active site architecture of CgMshA with other retaining glycosyltransferases suggests that the bent-back conformation of the donor molecule and the interaction of the ␤-phosphate with a positive charge (typically an arginine or metal ion) is a conserved feature of retaining glycosyltransferases (50,57).…”