Dataset Information

Side Chain Conformation Restriction in the Catalysis of Glycosidic Bond Formation by Leloir Glycosyltransferases, Glycoside Phosphorylases, and Transglycosidases.

ABSTRACT: Carbohydrate side chain conformation is an important factor in the control of reactivity at the anomeric center, ie, in the making and breaking of glycosidic bonds, whether chemically or, for hydrolysis, by glycoside hydrolases. In nature glycosidic bond formation is catalyzed out by glycosyltransferases (GTs), glycoside phosphoryases, and transglycosidases. By analysis of 118 crystal structures of sugar nucleotide dependent (Leloir) GTs, 136 crystal structures of glycoside phosphorylases, and 54 crystal structures of transglycosidases bound to hexopyranosides or their analogs at the donor site (-1 site), we determined that most enzymes that catalyze glycoside synthesis, be they GTs, glycoside phosphorylases or transglycosidases, restrict their substrate side chains to the most reactive gauche,gauche (gg) conformation to achieve maximum stabilization of the oxocarbenium ion-like transition state for glycosyl transfer. The galactose series deviates from this trend, with α-galactosyltransferases preferentially restricting their substrates to the second-most reactive gauche,trans (gt) conformation, and β-galactosyltransferases favoring the least reactive trans,gauche (tg) conformation. This insight will help progress the design and development of improved, conformationally-restricted GT inhibitors that take advantage of these inherent side chain preferences.

SUBMITTER: Quirke JCK

PROVIDER: S-EPMC8336929 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Publications

Side Chain Conformation Restriction in the Catalysis of Glycosidic Bond Formation by Leloir Glycosyltransferases, Glycoside Phosphorylases, and Transglycosidases.

Quirke Jonathan C K JCK Crich David D

ACS catalysis 20210413 9

Carbohydrate side chain conformation is an important factor in the control of reactivity at the anomeric center, ie, in the making and breaking of glycosidic bonds, whether chemically or, for hydrolysis, by glycoside hydrolases. In nature glycosidic bond formation is catalyzed out by glycosyltransferases (GTs), glycoside phosphoryases, and transglycosidases. By analysis of 118 crystal structures of sugar nucleotide dependent (Leloir) GTs, 136 crystal structures of glycoside phosphorylases, and 5 ...[more]

PMID: 34367723

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Project description:Sugar nucleotide-dependent (Leloir) glycosyltransferases from plants are important catalysts for the glycosylation of small molecules and natural products. Limitations on their applicability for biocatalytic synthesis arise because of low protein expression (≤10 mg/L culture) in standard microbial hosts. Here, we showed two representative glycosyltransferases: sucrose synthase from soybean and UGT71A15 from apple. A synthetic biology-based strategy of decoupling the enzyme expression from the Escherichia coli BL21(DE3) cell growth was effective in enhancing their individual (approximately fivefold) or combined (approximately twofold) production as correctly folded, biologically active proteins. The approach entails a synthetic host cell, which is able to shut down the production of host messenger RNA by inhibition of the E. coli RNA polymerase. Overexpression of the enzyme(s) of interest is induced by the orthogonal T7 RNA polymerase. Shutting down of the host RNA polymerase is achieved by l-arabinose-inducible expression of the T7 phage-derived Gp2 protein from a genome-integrated site. The glycosyltransferase genes are encoded on conventional pET-based expression plasmids that allow T7 RNA polymerase-driven inducible expression by isopropyl-β- d-galactoside. Laboratory batch and scaled-up (20 L) fed-batch bioreactor cultivations demonstrated improvements in an overall yield of active enzyme by up to 12-fold as a result of production under growth-decoupled conditions. In batch culture, sucrose synthase and UGT71A15 were obtained, respectively, at 115 and 2.30 U/g cell dry weight, corresponding to ∼5 and ∼1% of total intracellular protein. Fed-batch production gave sucrose synthase in a yield of 2,300 U/L of culture (830 mg protein/L). Analyzing the isolated glycosyltransferase, we showed that the improvement in the enzyme production was due to the enhancement of both yield (5.3-fold) and quality (2.3-fold) of the soluble sucrose synthase. Enzyme preparation from the decoupled production comprised an increased portion (61% compared with 26%) of the active sucrose synthase homotetramer. In summary, therefore, we showed that the expression in growth-arrested E. coli is promising for recombinant production of plant Leloir glycosyltransferases.

Dataset Information

Side Chain Conformation Restriction in the Catalysis of Glycosidic Bond Formation by Leloir Glycosyltransferases, Glycoside Phosphorylases, and Transglycosidases.

Publications

Side Chain Conformation Restriction in the Catalysis of Glycosidic Bond Formation by Leloir Glycosyltransferases, Glycoside Phosphorylases, and Transglycosidases.

Similar Datasets

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