Christiane Goedl

Sucrose phosphorylase: a powerful transglucosylation catalyst for synthesis of α-D-glucosides as industrial fine chemicals

Abstract Sucrose phosphorylase is a bacterial transglucosidase that catalyzes conversion of sucrose and phosphate into α-D-glucose-1-phosphate and D-fructose. The enzyme utilizes a glycoside hydrolase-like double displacement mechanism that involves a catalytically competent β-glucosyl enzyme intermediate. In addition to reaction with phosphate, glucosylated sucrose phosphorylase can undergo hydrolysis to yield α-D-glucose or it can decompose via glucosyl transfer to a hydroxy group in suitable acceptor molecules, giving new α-D-glucosidic products. The glucosyl acceptor specificity of sucrose phosphorylase is reviewed, focusing on applications of the enzyme in glucoside synthesis. Polyhydroxylated compounds such as sugars and sugar alcohols are often glucosylated efficiently. Aryl alcohols and different carboxylic acids also serve as acceptors for enzymatic transglucosylation. The natural osmolyte 2-O-(α-D-glucopyranosyl)-sn-glycerol (GG) was prepared by regioselective glucosylation of glycerol from sucrose using the phosphorylase from Leuconostoc mesenteroides. An industrial process for production of GG as active ingredient of cosmetic formulations has been recently developed. General advantages of sucrose phosphorylase as a transglucosylation catalyst lie in the use of sucrose as a high-energy glucosyl donor and the usually weak hydrolase activity of the enzyme towards substrate and product.

Glucosylglycerol and glucosylglycerate as enzyme stabilizers.

Compatible solutes constitute a diverse class of low‐molecular‐mass organic molecules that are accumulated in high intracellular concentrations in response to the external stress of hyperosmolality or high temperature. Many of these compounds like α, α‐trehalose are well known for their stabilizing effect on protein structure and could lead to development of more stable protein formulations. Negatively charged solutes like mannosylglycerate (R‐2‐O‐α‐D‐mannopyranosyl‐glycerate) are widespread among (hyper)thermophilic microorganisms and are thought to be exceptionally potent stabilizers of proteins under high‐temperature denaturation conditions. To further inquire into the role of compound charge for protective function, we have compared two naturally occurring and structurally related solutes, glucosylglycerol (2‐O‐α‐D‐glucopyranosyl‐sn‐glycerol) and glucosylglycerate (R‐2‐O‐α‐D‐glucopyranosyl‐glycerate), as stabilizers of different enzymes undergoing inactivation through elevated temperature or freeze drying, and benchmarked their effects against that of α,α‐trehalose. Glucosylglycerate in concentrations of ≥0.1 M was the most effective in preventing thermally induced loss of enzyme activity of lactate dehydrogenase, mannitol dehydrogenase, starch phosphorylase, and xylose reductase. α,α‐Trehalose could usually be replaced by glucosylglycerol without compromising enzyme stability. Glucosylglycerol and glucosylglycerate afforded substantial (eightfold) protection to mannitol dehydrogenase during freeze drying.

Single-step enzymatic synthesis of (R)-2-O-α-D-glucopyranosyl glycerate, a compatible solute from micro-organisms that functions as a protein stabiliser

Regioselective glucosylation of R-glycerate catalysed by sucrose phosphorylase in the presence of sucrose as the donor substrate provided the natural compatible solute (R)-2-O-alpha-D-glucopyranosyl glycerate with complete regioselectivity in an optimised synthetic yield of 90% R-glycerate converted and a concentration of about 270 mM.

Sucrose Phosphorylase Harbouring a Redesigned, Glycosyltransferase-Like Active Site Exhibits Retaining Glucosyl Transfer in the Absence of a Covalent Intermediate

Glycosyltransferases are key synthesis enzymes in glycobiology. Glycosyltransferases utilize an activated donor sugar substrate harbouring a substituted phosphate leaving group to bring about glycosidic bond formation in glycans, glycoconjugates and a wide variety of sugar-containing small biomolecules. Nucleophilic substitution at the anomeric carbon of the transferred glycosyl residue proceeds with either inversion or retention of configuration of the donor substrate (Scheme 1), and each stereochemical course necessitates a distinct catalytic mechanism. It is currently not clear how stereochemical control in glycosyltransferase-catalyzed group transfer is achieved. Inverting glycosyltransferases are proposed to utilize a direct-displacement SN2-like reaction mechanism in which departure of the leaving group is facilitated by a Lewis acid, and nucleophilic attack is assisted by a catalytic base of the enzyme (Scheme 1 A). Drawing analogy to glycoside hydrolases for which reaction coordinates leading to inversion or retention have been very well-characterized (see Scheme S1), a double displacement-like reaction involving a covalent glycosyl-enzyme intermediate appeared to be the preferred choice of mechanism for the retaining transferases. However, glycosyltransferase crystal structures revealed a surprising lack of conserved architecture in the region of the active site where the catalytic nucleophile would have to be positioned. In addition, despite exhaustive biochemical studies with techniques that have been successfully used in the characterization of ACHTUNGTRENNUNGretaining glycoside hydrolases, evidence supporting a covalent intermediate for retaining glycosyltransferases has remained elusive. An alternative mechanism, often referred to as internal return (SNi)-like [7] in the literature, was therefore considered (Scheme 1 B). 9] This is proposed to involve a short-lived ion pair intermediate, the formation of which arguably requires electrostatic front-side stabilization from the departing (substituted) phosphate and perhaps a certain amount of nucleophilic “push” from an enzyme group positioned on the backside of the glycosyl ring. 10] In addition to stabilizing the intermediate, hydrogen bonding between the incoming nucleophile and the leaving group would play an important basecatalytic role during activation of the acceptor hydroxyl group for nucleophilic attack. Suitable probes for direct interrogation of the glycosyltransferase mechanism in Scheme 1 B are not available. 9] We therefore chose a novel approach based on active site redesign of sucrose phosphorylase. This enzyme utilizes a glycoside hydrolase-like double-displacement mechanism to catalyze a glucosyltransferase-like reaction with retention of the a-anomeric configuration. a-d-Glucopyranosyl phosphate (aG1P) and dfructose are converted reversibly into sucrose (a-d-glucopyranosyl-1,2-b-d-fructofuranoside) and phosphate, however, phosphorolysis of sucrose is the physiological direction of the reACHTUNGTRENNUNGaction. Note that phosphorolysis is essentially the reverse of a glycosyltransferase reaction with nonsubstituted phosphate as the acceptor. Remodeling of sucrose phosphorylase from Leuconostoc mesenteroides involved site-directed replacements of the catalytic nucleophile (Asp196) and acid–base (Glu237) by the respective incompetent residues, Asn and Gln. This generated a doubly mutated enzyme, termed D196NE237Q, that resembled a typical glycosyltransferase with respect to the lack of catalytic site features to promote retaining glycosyl transfer. However, unlike glycosyltransferases, the relaxed specificity of the acceptor binding site of sucrose phosphorylase presented the essential advantage for the mechanistic characterization of D196N–E237Q. Figure 1 depicts the results of steady-state kinetic analysis of glucosylation of phosphate that was catalyzed by using purified preparations of wild-type enzyme and D196N–E237Q with a-d-glucopyranosyl fluoride (aG1F) as the donor substrate. ACHTUNGTRENNUNG lycosylfluorides are common alternative and highly reactive donor substrates for glycosyltransferases and glycoside hydrolases. In terms of catalytic efficiency (kcat/KM = 3.2 10 m 1 s ) and turnover number (kcat = 280 s ), wild-type sucrose phosphorylase utilized aG1F for phosphorolysis slightly more efficiently than it utilized sucrose. D196N–E237Q showed no activity towards sucrose as discussed later, and its kcat for reaction with aG1F was 10 -fold below the wild-type level. Data acquisition was carried out with an enzymatic assay that was absolutely selective for the a-anomer of d-glucopyranosyl phosphate; the assay clearly indicated that D196NE237Q promoted conversion of aG1F with retention of configuration. Formation of the product resulting from configurational inversion (b-d-glucopyranosyl phosphate) was rigorously excluded for both enzymes by using NMR spectroscopic characterization of the product mixture. Formation of aG1P did not occur in the absence of enzyme, and this ruled out uncatalyzed conversion of aG1F in an SNi-like retentive reaction such as that described by Sinnott and Jencks for trifluoroethanolysis of a-d-glucopyranosyl fluorides. Neither wild-type nor mutated enzymes accepted b-d-glucopyranosyl fluoride as donor [a] Dr. C. Goedl, Prof. Dr. B. Nidetzky Institute of Biotechnology and Biochemical Engineering Graz University of Technology Petersgasse 12/1, 8010 Graz (Austria) Fax: (+ 43) 316-873-8434 E-mail : bernd.nidetzky@tugraz.at Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900429.

Mechanistic differences among retaining disaccharide phosphorylases : insights from kinetic analysis of active site mutants of sucrose phosphorylase and α,α-trehalose phosphorylase

Sucrose phosphorylase utilizes a glycoside hydrolase-like double displacement mechanism to convert its disaccharide substrate and phosphate into alpha-d-glucose 1-phosphate and fructose. Site-directed mutagenesis was employed to characterize the proposed roles of Asp(196) and Glu(237) as catalytic nucleophile and acid-base, respectively, in the reaction of sucrose phosphorylase from Leuconostoc mesenteroides. The side chain of Asp(295) is suggested to facilitate the catalytic steps of glucosylation and deglucosylation of Asp(196) through a strong hydrogen bond (23 kJ/mol) with the 2-hydroxyl of the glucosyl oxocarbenium ion-like species believed to be formed in the transition states flanking the beta-glucosyl enzyme intermediate. An assortment of biochemical techniques used to examine the mechanism of alpha-retaining glucosyl transfer by Schizophyllum commune alpha,alpha-trehalose phosphorylase failed to provide evidence in support of a similar two-step catalytic reaction via a covalent intermediate. Mutagenesis studies suggested a putative active-site structure for this trehalose phosphorylase that is typical of retaining glycosyltransferases of fold family GT-B and markedly different from that of sucrose phosphorylase. While ambiguity remains regarding the chemical mechanism by which the trehalose phosphorylase functions, the two disaccharide phosphorylases have evolved strikingly different reaction coordinates to achieve catalytic efficiency and stereochemical control in their highly analogous substrate transformations.

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

The cDNA encoding trehalose phosphorylase, a family GT-4 glycosyltransferase from the fungus Schizophyllum commune, was isolated and expressed in Escherichia coli to yield functional recombinant protein in its full length of 737 amino acids. Unlike the natural phosphorylase that was previously obtained as a truncated 61 kDa monomer containing one tightly bound Mg2+, the intact enzyme produced in E. coli is a dimer and not associated with metal ions [Eis, Watkins, Prohaska and Nidetzky (2001) Biochem. J. 356, 757-767]. MS analysis of the slow spontaneous conversion of the full-length enzyme into a 61 kDa fragment that is fully active revealed that critical elements of catalysis and specificity of trehalose phosphorylase reside entirely in the C-terminal protein part. Intact and truncated phosphorylases thus show identical inhibition constants for the transition state analogue orthovanadate and alpha,alpha-trehalose (K(i) approximately 1 microM). Structure-based sequence comparison with retaining glycosyltransferases of fold family GT-B reveals a putative active centre of trehalose phosphorylase, and results of site-directed mutagenesis confirm the predicted crucial role of Asp379, His403, Arg507 and Lys512 in catalysis and also delineate a function of these residues in determining the large preference of the wild-type enzyme for the phosphorolysis compared with hydrolysis of alpha,alpha-trehalose. The pseudo-disaccharide validoxylamine A was identified as a strong inhibitor of trehalose phosphorylase (K(i)=1.7+/-0.2 microM) that displays 350-fold tighter binding to the enzyme-phosphate complex than the non-phosphorolysable substrate analogue alpha,alpha-thio-trehalose. Structural and electronic features of the inhibitor that may be responsible for high-affinity binding and their complementarity to an anticipated glucosyl oxocarbenium ion-like transition state are discussed.

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

Schizophyllum communeα,α‐trehalose phosphorylase utilizes a glycosyltransferase‐like catalytic mechanism to convert its disaccharide substrate into α‐d‐glucose 1‐phosphate and α‐d‐glucose. Recruitment of phosphate by the free enzyme induces α,α‐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 phosphate‐binding sub‐site. Loss of wild‐type catalytic efficiency for reaction with phosphate (kcat/Km = 21 000 m−1·s−1) was dramatic (≥107‐fold) in purified Arg507→Ala (R507A) and Lys512→Ala (K512A) enzymes, reflecting a corresponding change of comparable magnitude in kcat (Arg507) and Km (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 H2PO4−, 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 α,α‐trehalose (Ki = 0.4 μm) was completely disrupted in the R507A mutant but only weakened in the K512A mutant (Ki = 300 μm). Phosphate (50 mm) enhanced the basal hydrolase activity of the K512A mutant toward α,α‐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.

Studying non-covalent enzyme carbohydrate interactions by STD NMR

Saturation transfer difference NMR spectroscopy is used to study non-covalent interactions between four different glycostructure transforming enzymes and selected substrates and products. Resulting binding patterns represent a molecular basis of specific binding between ligands and biocatalysts. Substrate and product binding to Aspergillus fumigatus glycosidase and to Candida tenuis xylose reductase are determined under binding-only conditions. Measurement of STD effects in substrates and products over the course of enzymatic conversion provides additional information about ligand binding during reaction. Influences of co-substrates and co-enzymes in substrate binding are determined for Schizophyllum commune trehalose phosphorylase and C. tenuis xylose reductase, respectively. Differences between ligand binding to wild type enzyme and a corresponding mutant enzyme are shown for Corynebacterium callunae starch phosphorylase and its His-334-->Gly mutant. The resulting binding patterns are discussed with respect to the possibility that ligands do not only bind in the productive mode.