The structural biology of enzymes involved in natural product glycosylation

Singh, Shanteri; Phillips, George N.; Thorson, Jon S.

doi:10.1039/c2np20039b

Cited by 101 publications

(93 citation statements)

References 233 publications

(591 reference statements)

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“…Specifically, UDP/TDP-sugars (predominate in microbial natural product biosynthetic pathways) (5,20), CDP-sugars (important to the biosynthesis of pathogenic bacteria antigens) (21,22), GDP-sugars (used in a range of glycobiological processes) (5,23), and ADPsugars (important to intracellular trafficking, posttranslational modification of proteins, DNA repair, programmed cell death, and glycogen metabolism) (24)(25)(26) were targeted in this study. While UDP or TDP with 2-chloro-4-nitrophenyl glucoside were previously recognized as excellent substrates in the OleD-catalyzed sugar nucleotide syntheses reactions, ADP and GDP were identified as relatively poor substrates (i.e., seven-and 50-fold decrease in conversion for ADP and GDP, respectively, compared with equivalent reactions with UDP), while CDP was not recognized as a substrate (19).…”

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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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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“…1B). Specific glycosylation genes such as dehydratases, deoxygenases, dehydrogenases, methyltransferases, aminotransferases, and epimerases modify the common deoxysugar intermediate to yield the large diversity of sugar monomers in microbial GNPs (11,12). Phylogenetically, the highest GNP sugar diversity is found in actinobacteria (13).…”

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confidence: 99%

Glycogenomics as a mass spectrometry-guided genome-mining method for microbial glycosylated molecules

Kersten

Ziemert

Gonzalez³

et al. 2013

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

103

107

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Glycosyl groups are an essential mediator of molecular interactions in cells and on cellular surfaces. There are very few methods that directly relate sugar-containing molecules to their biosynthetic machineries. Here, we introduce glycogenomics as an experimentguided genome-mining approach for fast characterization of glycosylated natural products (GNPs) and their biosynthetic pathways from genome-sequenced microbes by targeting glycosyl groups in microbial metabolomes. Microbial GNPs consist of aglycone and glycosyl structure groups in which the sugar unit(s) are often critical for the GNP's bioactivity, e.g., by promoting binding to a target biomolecule. GNPs are a structurally diverse class of molecules with important pharmaceutical and agrochemical applications. Herein, O-and N-glycosyl groups are characterized in their sugar monomers by tandem mass spectrometry (MS) and matched to corresponding glycosylation genes in secondary metabolic pathways by a MS-glycogenetic code. The associated aglycone biosynthetic genes of the GNP genotype then classify the natural product to further guide structure elucidation. We highlight the glycogenomic strategy by the characterization of several bioactive glycosylated molecules and their gene clusters, including the anticancer agent cinerubin B from Streptomyces sp. SPB74 and an antibiotic, arenimycin B, from Salinispora arenicola CNB-527.secondary metabolite | drug discovery | microbial genomics | deoxysugar | polyketide

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“…This includes nonribosomal peptide (peptides that are not synthesized by ribosomes) synthetases (NRPS) and polyketide synthases (PKS) [199][200][201][202][203][204] required for the formation of the benzopyranopyrrole core unit. Other enzymes such as four halogenases [205] an O-methyltransferase [206][207][208] and an N-glycosyltransferase [209,210] necessary for further modifications of the core structure of pyralomicins have also been identified. In particular, the Nglycosyltransferase is involved in the transfer of either glucose or a pseudosugar (cyclitol) to the aglycone.…”

Section: Biosynthesis Of Pyralomicinsmentioning

confidence: 99%

Biosynthesis and Biological Activity of Carbasugars

Roscales

Plumet

2016

International Journal of Carbohydrate Chemistry

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The first synthesis of carbasugars, compounds in which the ring oxygen of a monosaccharide had been replaced by a methylene moiety, was described in 1966 by Professor G. E. McCasland's group. Seven years later, the first true natural carbasugar (5a-carba-R-D-galactopyranose) was isolated from a fermentation broth of Streptomyces sp. MA-4145. In the following decades, the chemistry and biology of carbasugars have been extensively studied. Most of these compounds show interesting biological properties, especially enzymatic inhibitory activities, and, in consequence, an important number of analogues have also been prepared in the search for improved biological activities. The aim of this review is to give coverage on the progress made in two important aspects of these compounds: the elucidation of their biosynthesis and the consideration of their biological properties, including the extensively studied carbapyranoses as well as the much less studied carbafuranoses.

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The structural biology of enzymes involved in natural product glycosylation

Cited by 101 publications

References 233 publications

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

Glycogenomics as a mass spectrometry-guided genome-mining method for microbial glycosylated molecules

Biosynthesis and Biological Activity of Carbasugars

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