Engineered Urdamycin Glycosyltransferases Are Broadened and Altered in Substrate Specificity

Hoffmeister, Dirk; Wilkinson, Barrie; Foster, Graham; Sidebottom, Philip J.; Ichinose, Koji; Bechthold, Andreas

doi:10.1016/s1074-5521(02)00114-x

Cited by 87 publications

(55 citation statements)

References 39 publications

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“…This observation implies that the overall GT structure provides a general catalytic platform and that the GT chimeras produced by swapping the N3 and N5 regions might contribute to changes in the acceptor regiospecificity and increased reaction promiscuity. This contention is further supported by prior mutagenesis studies that implicate the N3 and N5 loops as influencing reaction specificity (26)(27)(28).…”

Section: Discussionmentioning

confidence: 57%

Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity

Chang

Singh

Helmich

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Glycosyltransferases are useful synthetic catalysts for generating natural products with sugar moieties. Although several natural product glycosyltransferase structures have been reported, design principles of glycosyltransferase engineering for the generation of glycodiversified natural products has fallen short of its promise, partly due to a lack of understanding of the relationship between structure and function. Here, we report structures of all four calicheamicin glycosyltransferases (CalG1, CalG2, CalG3, and CalG4), whose catalytic functions are clearly regiospecific. Comparison of these four structures reveals a conserved sugar donor binding motif and the principles of acceptor binding region reshaping. Among them, CalG2 possesses a unique catalytic motif for glycosylation of hydroxylamine. Multiple glycosyltransferase structures in a single natural product biosynthetic pathway are a valuable resource for understanding regiospecific reactions and substrate selectivities and will help future glycosyltransferase engineering.

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Section: Discussionmentioning

confidence: 57%

Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity

Chang

Singh

Helmich

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Within the reported OleD crystal structure (27), residue 67 is situated amid a loop region (amino acids 60-76, loop N3), which is hypervariable in other GTs possessing the GT-B fold and contributes to forming the "donor" site in the context of GT-catalyzed reverse reactions (14,31). Studies with other GT-B fold GTs have highlighted additional positions within the N3 loop as impacting upon enzyme specificity (32).…”

Section: Resultsmentioning

confidence: 99%

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

Gantt

Peltier‐Pain

Singh

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in k cat /K m of >400-fold and >15-fold for formation of NDP-glucoses and UDP-sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP-sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.carbohydrate | enzyme | glycobiology | protein engineering T he lack of accessibility and availability of uncommon and uniquely functionalized sugar nucleotides (NDP-sugars) continues to restrict research focused upon understanding the regulation, biosynthesis, and/or role of glycosylated macromolecules and glycosylated small molecules in biology or therapeutic development (1-7). Although there are many reported chemical, enzymatic, and chemoenzymatic strategies for NDP-sugar synthesis, those that extend beyond the reach of common biological sugars (e.g., Dglucose, D-galactose, etc.) nearly all suffer from long reaction times (>16 h), relatively low yields, and difficulties associated with product purification and/or stability (3,4,8,9). Thus, the development of robust methods for sugar nucleotide synthesis directly compatible to the downstream biological processes to be studied may be advantageous.From a traditional viewpoint, NDP-sugars are used as donors by Leloir glycosyltransferases (sugar nucleotide-dependent enzymes) for formation of glycosidic bonds. However, many glycosyltransferase (GT)-catalyzed reactions are known to be readily reversible, enabling the "pirating" of unique sugars from natural products or alternative donors (resulting in generation of the respective sugar nucleotide) and one-pot sugar exchange reactions between unique natural products (4, 10-13). This general reaction feature, in conjunction with availability of highly permissive glycosyltransferases (14-18) and simple donors designed to fundamentally alter the reaction thermodynamics, recently enabled a unique platform for NDP-sugar synthesis and a high throughput colorimetric screen for NDP-sugar formation and utilization (19). While the prior platform proof-of-concept study highlighted the syntheses of 22 natural and nonnatural TDP/UDP-sugars from 11 distinct 2-chloro-4-nitrophenyl glycoside donors using a single GT catalyst (Fig. 1A) (19), the substrate specificity of the glycosyltransferase used ...

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“…However, as yet the determinants of this specificity are poorly understood. This lack of understanding presents challenges to the interpretation of substrate activity data (6,7), GT rational redesign (8,9), and activity prediction (10,11). Future progress in predicting GT sequence-structure-activity relationships will depend on a greater number of studies that characterize the key determinants of activity and specificity.…”

mentioning

confidence: 99%

A Kinetic Analysis of Regiospecific Glucosylation by Two Glycosyltransferases of Arabidopsis thaliana

Cartwright

Lim

Kleanthous

et al. 2008

Journal of Biological Chemistry

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Plant Family 1 glycosyltransferases (GTs) recognize a wide range of natural and non-natural scaffolds and have considerable potential as biocatalysts for the synthesis of small molecule glycosides. Regiospecificity of glycosylation is an important property, given that many acceptors have multiple potential glycosylation sites. This study has used a domain-swapping approach to explore the determinants of regiospecific glycosylation of two GTs of Arabidopsis thaliana, UGT74F1 and UGT74F2. The flavonoid quercetin was used as a model acceptor, providing five potential sites for O-glycosylation by the two GTs. As is commonly found for many plant GTs, both of these enzymes produce distinct multiple glycosides of quercetin. A high performance liquid chromatography method has been established to perform detailed steady-state kinetic analyses of these concurrent reactions. These data show the influence of each parameter in determining a GT product formation profile toward quercetin. Interestingly, construction and kinetic analyses of a series of UGT74F1/F2 chimeras have revealed that mutating a single amino acid distal to the active site, Asn-142, can lead to the development of a new GT with a more constrained regiospecificity. This ability to form the 4-O-glucoside of quercetin is transferable to other flavonoid scaffolds and provides a basis for preparative scale production of flavonoid 4-O-glucosides through the use of wholecell biocatalysis.The glycosylation of small hydrophobic molecules is known to alter their chemical and biological characteristics. The alterations include changes in water solubility, stability, pharmacokinetic properties, and bioactivity (1, 2). Enzymes that catalyze these reactions are Family 1 glycosyltransferases (GTs), 3 with representatives found in a wide range of prokaryotic and eukaryotic organisms.Extensive study of GT activities has demonstrated that the enzymes display an exquisite regio-(3), enantio-(4), and chemospecificity (5) toward the acceptor molecule. However, as yet the determinants of this specificity are poorly understood. This lack of understanding presents challenges to the interpretation of substrate activity data (6, 7), GT rational redesign (8, 9), and activity prediction (10, 11). Future progress in predicting GT sequence-structure-activity relationships will depend on a greater number of studies that characterize the key determinants of activity and specificity.Quercetin is the most abundant flavonoid in the human diet (12) and has frequently been used as a model substrate for GT activity (13-15). Quercetin aglycone is found only at low concentrations in the primary dietary source, plants, where glycosylated forms predominate (16). In addition, in mammals, glycosides are the major byproducts of quercetin phase II metabolism (17). Many studies have shown that glycosylation of quercetin significantly affects bioavailability and efficacy with respect to anti-oxidant, anti-proliferative, and anti-cancer properties (18). There are also some data to indicate that the positi...

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Engineered Urdamycin Glycosyltransferases Are Broadened and Altered in Substrate Specificity

Cited by 87 publications

References 39 publications

Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity

Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

A Kinetic Analysis of Regiospecific Glucosylation by Two Glycosyltransferases of Arabidopsis thaliana

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