Exploiting the Reversibility of Natural Product Glycosyltransferase-Catalyzed Reactions

Zhang, Changsheng; Griffith, Byron R.; Fu, Qiang; Albermann, Christoph; Fu, Xun; Lee, In‐Kyoung; Li, Lingjun; Thorson, Jon S.

doi:10.1126/science.1130028

Cited by 263 publications

(210 citation statements)

References 24 publications

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“…Based upon this assessment, seven putative sugar donor-interacting positions (amino acids 67, 74, 85, 111-113, and 134) and eight potential NDP acceptor-interacting positions (amino acids 132, 242, 243, 268, 290, 309, 330, and 331) were selected for saturation mutagenesis to present the potential of 285 possible mutants to be generated for screening with each desired substrate pairing. Of these positions, five (amino acids 67, 112, 132, 242, and 268) were previously identified as impacting upon OleD permissivity and/or proficiency (9)(10)(11)(12)(13)(14). Special care was taken to ensure that the substrate binding pockets for both the putative 2-chloro-4-nitrophenyl glycoside donors (donors 1 and 4-8) and NDP acceptors were equally represented ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The impressive aglycon malleability of OleD (17)(18)(19), the demonstrated ability to expand the sugar scope of this catalyst, and the demonstrated superior ability of Loki to also catalyze "forward" coupled transglycosylation reactions suggest a range of exciting opportunities. Examples include extending applications toward (i) modified nucleoside-based NDPs (3) (reminiscent of the kinase "bump and hole" strategies) (38); (ii) specific sugardrug or sugar-natural product pairings (39)(40)(41); (iii) a broadened scope of deoxy, dideoxy, and/or uniquely functionalized sugars (e.g., sugars bearing amino-, N-akyl, O-alkyl, C-alkyl-, nitro, nitroso-, thio-modifications) (2,4,7,10,15,18,19); and (iv) other important biomolecules (e.g., proteins, polysaccharides) (42)(43)(44)(45).…”

Section: Preliminary Assessment Of Loki In a Model Coupled Forward Rementioning

confidence: 99%

“…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)(11)(12)(13). This general reaction feature, in conjunction with availability of highly permissive glycosyltransferases (14)(15)(16)(17)(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).…”

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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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Section: Resultsmentioning

confidence: 99%

Section: Preliminary Assessment Of Loki In a Model Coupled Forward Rementioning

confidence: 99%

mentioning

confidence: 99%

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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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“…51) Very recently, typical GT enzymes using NDP sugars are also shown to catalyze reversible reactions. 52) However, phosphorylases still have advantages for practical use, with their higher stability and lower cost substrates. The first sugar phosphorylase whose 3 D structure was determined was that of GT35 glycogen phosphorylase.…”

Section: Reclassification From Gt36 To Gh94: Gh-type In-mentioning

confidence: 99%

New Structural Insights on Carbohydrate-active Enzymes

Fushinobu¹,

Hidaka²,

Miyanaga³

et al. 2007

J. Appl. Glycosci.

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Polysaccharides show a huge degree of diversity owing to their variety of sugar components (aldoses, ketoses and their stereoisomers), the variety of glycosidic bonds (e.g., α 1,4 , α 1,6 , β 1,4 , β 1,3 , etc.), different degrees of polymerization, and different degrees of branching. Therefore, they can be involved in many types of biological function with distinct physicochemical characteristics. It is especially intriguing that α and β glucans, such as amylose (starch) and cellulose, are built from the same glucose units, differing only in the anomeric configurations of glycosidic bonds. Enzymes that hydrolyze these glucans, amylases and cellulases, are the two largest groups of glycoside hydrolases, and have been studied for a long time. A framework to discuss a whole variety of glycoside hydrolases with their three dimensional structural aspects has been established within the last 15 years. In the late 1980s, Henrissat et al. began classification of the amino acid sequences of enzymes, focusing on cellulases and hemicellulases. 1) At the earliest stage of their classification, the α amylase family was treated as belonging to a separate system.2) In 1991, the naming system of the families changed from alphabetical to numerical, and the system expanded to 35 "glycosyl hydrolase" families, incorporating three well known amylase families, GH13 (retaining α amylase family), GH14 (inverting β amylase family), and GH15 (inverting glucoamylase family), as well as many other enzymes.3) All enzymes cleaving, modifying, and creating glycosidic bonds, and modules binding to carbohydrates have been incorporated into the CAZy database (http: www.cazy.org CAZY ). Glycoside hydrolase (GH) families now include enzymes that hydrolyze the glycosidic bonds between two or more carbohydrates or between a carbohydrate and a non carbohydrate moiety. At present, there are 101 families in the GH class ( Fig. 1; note, five have been deleted or reassigned to other classes), and it will continue to grow.The first 3 D structure of a glycoside hydrolase to be determined was that of hen egg white lysozyme (classified into GH22) in the mid 1960s.4) Even in the early 1990s, however, only a limited number of GH enzyme structures were available. Henrissat and colleagues took amino acid sequence information as the primary basis for classification, but they also used hydrophobic cluster analysis from the earliest stage, trying to incorporate as much 3 D structure based information as possible.5) The significant advances in structural studies of GH enzymes over the last decade have indicated that these enzymes show a vast array of tertiary scaffolds, described as "a marketplace of different structures". 6)Our group has been trying to solve crystal structures of useful enzymes for use in bioindustry. Two of these were the first crystal structures within the GH42 and GH57 families, 7,8) and we then began to focus on CAZy enzymes belonging to structurally unknown families. We have since solved two more GH families, and provided the first crystal s...

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“…Thus far, only relatively few Gtfs have been characterized in vitro and at the mechanistic level, considering the vast number of Gtf sequences revealed in the data base (9,(23)(24)(25)(27)(28)(29)(30)(31)(32)(33)(34). Especially, SpnG is involved in the biosynthetic construction of unique multicyclic or highly modified PK-I glycoside (4,5).…”

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confidence: 99%

Functional Characterization and Substrate Specificity of Spinosyn Rhamnosyltransferase by in Vitro Reconstitution of Spinosyn Biosynthetic Enzymes

Chen

Lin

et al. 2009

Journal of Biological Chemistry

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Spinosyn, a potent insecticide, is a novel tetracyclic polyketide decorated with D-forosamine and tri-O-methyl-L-rhamnose. Spinosyn rhamnosyltransferase (SpnG) is a key biocatalyst with unique sequence identity and controls the biosynthetic maturation of spinosyn. The rhamnose is critical for the spinosyn insecticidal activity and cell wall biosynthesis of the spinosyn producer, Saccharopolyspora spinosa. In this study, we have functionally expressed and characterized SpnG and the three enzymes, Gdh, Epi, and Kre, responsible for dTDP-L-rhamnose biosynthesis in S. spinosa by purified enzymes from Escherichia coli. Most notably, the substrate specificity of SpnG was thoroughly characterized by kinetic and inhibition experiments using various NDP sugar analogs made by an in situ combination of NDP-sugar-modifying enzymes. SpnG was found to exhibit striking substrate promiscuity, yielding corresponding glycosylated variants. Moreover, the critical residues presumably involved in catalytic mechanism of Gdh and SpnG were functionally evaluated by site-directed mutagenesis. The information gained from this study has provided important insight into molecular recognition and mechanism of the enzymes, especially SpnG. The results have made possible the structureactivity characterization of SpnG, as well as the use of SpnG or its engineered form to serve as a combinatorial tool to make spinosyn analogs with altered biological activities and potency.Spinosad, a mixture of spinosyns A and D (ϳ85 and 15%, respectively) produced by the actinomycete Saccharopolyspora spinosa, has been proven highly effective against many chewing insect pests and designated as a reduced risk pesticide excellent in both environmental and mammalian toxicological considerations (1). Spinosyns have thus attracted increasing efforts on structural modifications to improve their insecticidal activity as well as to prevent possible resistance problems (2, 3). Structurally, spinosyns are novel glycosylated macrolides consisting of a 21-carbon tetracyclic lactone decorated with tri-O-methyl-Lrhamnose and D-forosamine (see Scheme 1). The tetracyclic aglycone scaffold is delicately constructed first by type I polyketide (PK-I) 2 synthases, followed by proposed postmodification events, including FAD-oxidation at C-15, Michael addition between C-3 and C-14, ␥-dehydration at C-11, and Diels-Alder cyclization (4, 5). The spinosyn biosynthesis genes recently cloned and sequenced are located in an ϳ80-kb cluster of the S. spinosa genome, except that the four genes involved in L-rhamnose (L-Rha) biosynthesis, gtt (NDP-glucose synthase), gdh (NDP-glucose 4,6-dehydratase), epi (NDP-4-keto-6-deoxyglucose epimerase), and kre (NDP-4-ketorhamnose reductase), are located in three different regions of the genome (6). Previous genetic experiments have suggested that the polyketide aglycone is biosynthesized prior to immediate attachment of L-Rha by the rhamnosyltransferase SpnG, followed by consecutive tri-O-methylation of the L-Rha on resulting spinosyn rhamnosyl pseudoa...

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Exploiting the Reversibility of Natural Product Glycosyltransferase-Catalyzed Reactions

Cited by 263 publications

References 24 publications

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

New Structural Insights on Carbohydrate-active Enzymes

Functional Characterization and Substrate Specificity of Spinosyn Rhamnosyltransferase by in Vitro Reconstitution of Spinosyn Biosynthetic Enzymes

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