Aromatic-Mediated Carbohydrate Recognition in Processive Serratia marcescens Chitinases.

Microorganisms use a host of enzymes, including processive glycoside hydrolases, to deconstruct recalcitrant polysaccharides to sugars. Processive glycoside hydrolases closely associate with polymer chains and repeatedly cleave glycosidic linkages without dissociating from the crystalline surface after each hydrolytic step; they are typically the most abundant enzymes in both natural secretomes and industrial cocktails by virtue of their significant hydrolytic potential. The ubiquity of aromatic residues lining the enzyme catalytic tunnels and clefts is a notable feature of processive glycoside hydrolases. We hypothesized that these aromatic residues have uniquely defined roles, such as substrate chain acquisition and binding in the catalytic tunnel, that are defined by their local environment and position relative to the substrate and the catalytic center. Here, we investigated this hypothesis with variants of Serratia marcescens family 18 processive chitinases ChiA and ChiB. We applied molecular simulation and free energy calculations to assess active site dynamics and ligand binding free energies. Isothermal titration calorimetry provided further insight into enthalpic and entropic contributions to ligand binding free energy. Thus, the roles of six aromatic residues, Trp-167, Trp-275, and Phe-396 in ChiA, and Trp-97, Trp-220, and Phe-190 in ChiB, have been examined. We observed that point mutation of the tryptophan residues to alanine results in unfavorable changes in the free energy of binding relative to wild-type. The most drastic effects were observed for residues positioned at the "entrances" of the deep substrate-binding clefts and known to be important for processivity. Interestingly, phenylalanine mutations in ChiA and ChiB had little to no effect on chito-oligomer binding, in accordance with the limited effects of their removal on chitinase functionality.

Diastereoselective Synthesis of Glycosyl Phosphates by Using a Phosphorylase-Phosphatase Combination Catalyst.

Sugar phosphates play an important role in metabolism and signaling, but also as constituents of macromolecular structures. Selective phosphorylation of sugars is chemically difficult, particularly at the anomeric center. We report phosphatase-catalyzed diastereoselective "anomeric" phosphorylation of various aldose substrates with α-D-glucose 1-phosphate, derived from phosphorylase-catalyzed conversion of sucrose and inorganic phosphate, as the phosphoryl donor. Simultaneous and sequential two-step transformations by the phosphorylase-phosphatase combination catalyst yielded glycosyl phosphates of defined anomeric configuration in yields of up to 70 % based on the phosphate applied to the reaction. An efficient enzyme-assisted purification of the glycosyl phosphate products from reaction mixtures was established.

Interplay of catalytic subsite residues in the positioning of α-d-glucose 1-phosphate in sucrose phosphorylase.

Kinetic and molecular docking studies were performed to characterize the binding of α-d-glucose 1-phosphate (αGlc 1-P) at the catalytic subsite of a family GH-13 sucrose phosphorylase (from L. mesenteroides) in wild-type and mutated form. The best-fit binding mode of αGlc 1-P dianion had the phosphate group placed anti relative to the glucosyl moiety (adopting a relaxed 4C1 chair conformation) and was stabilized mainly by hydrogen bonds from residues of the enzyme׳s catalytic triad (Asp196, Glu237 and Asp295) and from Arg137. Additional feature of the αGlc 1-P docking pose was an intramolecular hydrogen bond (2.7 Å) between the glucosyl C2-hydroxyl and the phosphate oxygen. An inactive phosphonate analog of αGlc 1-P did not show binding to sucrose phosphorylase in different experimental assays (saturation transfer difference NMR, steady-state reversible inhibition), consistent with evidence from molecular docking study that also suggested a completely different and strongly disfavored binding mode of the analog as compared to αGlc 1-P. Molecular docking results also support kinetic data in showing that mutation of Phe52, a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase. However, when combined with a second mutation involving one of the catalytic triad residues, the mutation of Phe52 by Ala caused complete (F52A_D196A; F52A_E237A) or very large (F52A_D295A) disruption of the proposed productive binding mode of αGlc 1-P with consequent effects on the enzyme activity. Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp196) are suggested. High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.

Phosphoryl transfer from α-d-glucose 1-phosphate catalyzed by Escherichia coli sugar-phosphate phosphatases of two protein superfamily types.

The Cori ester α-d-glucose 1-phosphate (αGlc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes αGlc 1-P for flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-phosphate phosphatases from Escherichia coli were characterized in this study for utilization of αGlc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic αGlc 1-P phosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (in E. coli Origami B) gave a functional enzyme preparation (kcat for phosphoryl transfer from αGlc 1-P to water, 40 s(-1)) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys(189) and Cys(195). Enzymatic phosphoryl transfer from αGlc 1-P to water in H2 (18)O solvent proceeded with complete (18)O label incorporation into the phosphate released, consistent with catalytic reaction through O-1-P, but not C-1-O, bond cleavage. Hydrolase activity of both enzymes was not restricted to a glycosidic phosphomonoester substrate, and d-glucose 6-phosphate was converted with a kcat similar to that of αGlc 1-P. By examining phosphoryl transfer from αGlc 1-P to an acceptor substrate other than water (d-fructose or d-glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized αGlc 1-P mainly for phosphoryl transfer to water. By applying d-fructose in 10-fold molar excess over αGlc 1-P (20 mM), enzymatic conversion furnished d-fructose 1-phosphate as the main product in a 55% overall yield. Agp is a promising biocatalyst for use in transphosphorylation from αGlc 1-P.

Yihx-encoded haloacid dehalogenase-like phosphatase HAD4 from Escherichia coli is a specific α-d-glucose 1-phosphate hydrolase useful for substrate-selective sugar phosphate transformations.

Phosphomonoester hydrolases (phosphatases; EC 3.1.3.) often exhibit extremely relaxed substrate specificity which limits their application to substrate-selective biotransformations. In search of a phosphatase catalyst specific for hydrolyzing α-d-glucose 1-phosphate (αGlc 1-P), we selected haloacid dehalogenase-like phosphatase 4 (HAD4) from Escherichia coli and obtained highly active recombinant enzyme through a fusion protein (Zbasic2_HAD4) that contained Zbasic2, a strongly positively charged three α-helical bundle module, at its N-terminus. Highly pure Zbasic2_HAD4 was prepared directly from E. coli cell extract using capture and polishing combined in a single step of cation exchange chromatography. Kinetic studies showed Zbasic2_HAD4 to exhibit 565-fold preference for hydrolyzing αGlc 1-P (kcat/KM = 1.87 ± 0.03 mM-1 s-1; 37 °C, pH 7.0) as compared to d-glucose 6-phosphate (Glc 6-P). Also among other sugar phosphates, αGlc 1-P was clearly preferred. Using different mixtures of αGlc 1-P and Glc 6-P (e.g. 180 mM each) as the substrate, Zbasic2_HAD4 could be used to selectively convert the αGlc 1-P present, leaving back all of the Glc 6-P for recovery. Zbasic2_HAD4 was immobilized conveniently using direct loading of E. coli cell extract on sulfonic acid group-containing porous carriers, yielding a recyclable heterogeneous biocatalyst that was nearly as effective as the soluble enzyme, probably because protein attachment to the anionic surface occurred in a preferred orientation via the cationic Zbasic2 module. Selective removal of αGlc 1-P from sugar phosphate preparations could be an interesting application of Zbasic2_HAD4 for which readily available broad-spectrum phosphatases are unsuitable.

Chiral resolution through stereoselective transglycosylation by sucrose phosphorylase: application to the synthesis of a new biomimetic compatible solute, (R)-2-O-α-D-glucopyranosyl glyceric acid amide.

Sucrose phosphorylase catalysed glycosylation of glyceric acid amide with complete regio- and diastereo-selectivity is studied. (R)-2-O-α-D-Glucopyranosyl glyceric acid amide was obtained in high yield from single-step transformation of racemic glyceric acid amide and sucrose. Non-productive binding of (S)-glyceric acid amide appeared to underlie strict enantiodiscrimination by the enzyme, thus supporting chiral resolutions based on stereoselective transglycosylation.

Examining the role of phosphate in glycosyl transfer reactions of Cellulomonas uda cellobiose phosphorylase using D-glucal as donor substrate.

Cellobiose phosphorylase from Cellulomonas uda (CuCPase) is shown to utilize D-glucal as slow alternative donor substrate for stereospecific glycosyl transfer to inorganic phosphate, giving 2-deoxy-α-D-glucose 1-phosphate as the product. When performed in D(2)O, enzymatic phosphorolysis of D-glucal proceeds with incorporation of deuterium in equatorial position at C-2, implying a stereochemical course of reaction where substrate becomes protonated from below its six-membered ring through stereoselective re side attack at C-2. The proposed catalytic mechanism, which is supported by results of docking studies, involves direct protonation of D-glucal by the enzyme-bound phosphate, which then performs nucleophilic attack on the reactive C-1 of donor substrate. When offered D-glucose next to D-glucal and phosphate, CuCPase produces 2-deoxy-ß-D-glucosyl-(1→4)-D-glucose and 2-deoxy-α-D-glucose 1-phosphate in a ratio governed by mass action of the two acceptor substrates present. Enzymatic synthesis of 2-deoxy-ß-D-glucosyl-(1→4)-D-glucose is effectively promoted by catalytic concentrations of phosphate, suggesting that catalytic reaction proceeds through a quaternary complex of CuCPase, D-glucal, phosphate, and D-glucose. Conversion of D-glucal and phosphate presents a convenient single-step synthesis of 2-deoxy-α-D-glucose 1-phosphate that is difficult to prepare chemically.

Aromatic interactions at the catalytic subsite of sucrose phosphorylase: their roles in enzymatic glucosyl transfer probed with Phe52→Ala and Phe52→Asn mutants.

Mutants of Leuconostoc mesenteroides sucrose phosphorylase having active-site Phe(52) replaced by Ala (F52A) or Asn (F52N) were characterized by free energy profile analysis for catalytic glucosyl transfer from sucrose to phosphate. Despite large destabilization (≥3.5kcal/mol) of the transition states for enzyme glucosylation and deglucosylation in both mutants as compared to wild-type, the relative stability of the glucosyl enzyme intermediate was weakly affected by substitution of Phe(52). In reverse reaction where fructose becomes glucocylated, "error hydrolysis" was the preponderant path of breakdown of the covalent intermediate of F52A and F52N. It is proposed, therefore, that Phe(52) facilitates reaction of the phosphorylase through (1) positioning of the transferred glucosyl moiety at the catalytic subsite and (2) strong cation-π stabilization of the oxocarbenium ion-like transition states flanking the covalent enzyme intermediate.

Regioselective O-glucosylation by sucrose phosphorylase: a promising route for functional diversification of a range of 1,2-propanediols.

1,2-Propanediol and 3-aryloxy/alkyloxy derivatives thereof are bulk commodities produced directly from glycerol. Glycosylation is a promising route for their functional diversification into useful fine chemicals. Regioselective glucosylation of the secondary hydroxyl in different 1,2-propanediols was achieved by a sucrose phosphorylase-catalyzed transfer reaction where sucrose is the substrate and 2-O-alpha-d-glucopyranosyl products are exclusively obtained. Systematic investigation for optimization of the biocatalytic synthesis included prevention of sucrose hydrolysis, which occurs in the process as a side reaction of the phosphorylase. In addition to 'nonproductive' depletion of donor substrate, the hydrolysis also resulted in formation of maltose and kojibiose (up to 45%) due to secondary enzymatic glucosylation of the glucose thus produced. Using 3-ethoxy-1,2-propanediol as the acceptor substrate (1.0M), the desired transfer product was obtained in about 65% yield when employing a moderate (1.5-fold) excess of sucrose donor. Loss of the glucosyl substrate to 'glucobiose' by-products was minimal (<7.5%) under these conditions. The reactivity of other acceptors decreased in the order, 3-methoxy-1,2-propanediol>1,2-propanediol>3-allyloxy-1,2-propanediol>3-(o-methoxyphenoxy)-1,2-propanediol>3-tert-butoxy-1,2-propanediol. Glucosylated 1,2-propanediols were not detectably hydrolyzed by sucrose phosphorylase so that their synthesis by transglucosylation occurred simply under quasi-equilibrium reaction conditions.