Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases

Hu, Yanan; Chen, Lan; Ha, Sha; Gross, Ben; Falcone, Brian V.; Walker, Deborah; Mokhtarzadeh, Maryam; Walker, Suzanne

doi:10.1073/pnas.0235749100

Cited by 218 publications

(257 citation statements)

References 35 publications

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“…This result is, again, in good agreement with the concluded role of NDP on SpnG. In contrast, previous studies reported that MurG accepted only an UDP-sugar, and vancomycin group Gtfs utilized both UDPand TDP-sugars (26,40). For the discrimination between UDPsugar and TDP-sugar, a Glu residue was observed in MurG to hydrogen-bond to the ribose 2Ј-and 3Ј-hydroxyls, whereas in vancomycin group Gtfs a replacement with Val or Leu was found (25), forming no hydrogen bond.…”

Section: Discussionsupporting

confidence: 72%

“…Ribbon representation of ␣␤␣ motif in MurG, GtfA, GtfB, GtfD, and SpnG. The ␣␤␣ motif structures of MurG, GtfA, GtfB, and GtfD are known as reported (23)(24)(25)(26), whereas that of SpnG was generated in this study by SWISS-MODEL with GtfB as a template (43) (available on the World Wide Web). The conserved ␣␤␣ motif is proposed to be involved in the binding of NDP-sugar and exhibits similar folding in these five glycosyltransferases.…”

Section: Discussionmentioning

confidence: 99%

“…8). However, the analysis revealed that SpnG retained the ␣␤␣ motif previously proposed to participate in the Gtf catalysis and NDP-sugar binding (23)(24)(25)(26), as illustrated by our homology modeling in Fig. 9 and secondary structure prediction in Fig.…”

mentioning

confidence: 99%

“…S2), some candidates, including Thr-297, Thr-300, and Asp-319, could be speculated to participate in dTDP-sugar binding in SpnG (25,26). The Thr-300 equivalent in MurG, Glu-269, was previously reported to contact both 2Ј-and 3Ј-ribose hydroxyls of UDP to discriminate against dTDP (26). Hence, SpnG was engineered by replacing Thr-300 with Asp and Val to examine its functional role and, if possible, to accept UDP-sugar.…”

mentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…The emergence of resistance to ␤-lactams and other clinically used antibiotics has prompted intense interest in understanding peptidoglycan biosynthesis in detail. Major advances have been made in recent years with respect to the characterization of key biosynthetic enzymes (3)(4)(5). Several structures have been reported for enzymes that catalyze transpeptidation (6,7), but no PGT structures have been described.…”

mentioning

confidence: 99%

Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis

Yuan

Barrett²,

Zhang³

et al. 2007

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

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122

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Peptidoglycan is an essential polymer that forms a protective shell around bacterial cell membranes. Peptidoglycan biosynthesis is the target of many clinically used antibiotics, including the ␤-lactams, imipenems, cephalosporins, and glycopeptides. Resistance to these and other antibiotics has prompted interest in an atomic-level understanding of the enzymes that make peptidoglycan. Representative structures have been reported for most of the enzymes in the pathway. Until now, however, there have been no structures of any peptidoglycan glycosyltransferases (also known as transglycosylases), which catalyze formation of the carbohydrate chains of peptidoglycan from disaccharide subunits on the bacterial cell surface. We report here the 2.1-Å crystal structure of the peptidoglycan glycosyltransferase (PGT) domain of Aquifex aeolicus PBP1A. The structure has a different fold from all other glycosyltransferase structures reported to date, but it bears some resemblance to -lysozyme, an enzyme that degrades the carbohydrate chains of peptidoglycan. An analysis of the structure, combined with biochemical information showing that these enzymes are processive, suggests a model for glycan chain polymerization.antibiotic resistance ͉ penicillin-binding protein ͉ cell wall ͉ transglycosylase T he major component of the bacterial cell wall is a cross-linked glycopeptide polymer called peptidoglycan. This polymer surrounds the cytoplasmic membrane of bacteria and functions as an exoskeleton, maintaining cell shape and stabilizing the membrane against fluctuations in osmotic pressure. Peptidoglycan is synthesized in an intracellular phase in which UDP-Nacetylglucosamine is converted to a diphospholipid-linked disaccharide-pentapeptide known as Lipid II, and in an extracellular phase in which the disaccharide (NAG-NAM) subunits of translocated Lipid II are coupled by peptidoglycan glycosyltransferases (PGTs; also known as transglycosylases) to form linear carbohydrate chains ( Fig. 1), which are cross-linked through the attached peptide moieties by transpeptidases (1). A functioning peptidoglycan pathway is required for bacterial cell growth and division, and compounds that inhibit peptidoglycan biosynthesis have antibiotic activity (2). For example, the ␤-lactams, which have been used for decades to treat bacterial infections, irreversibly inhibit the transpeptidases. The emergence of resistance to ␤-lactams and other clinically used antibiotics has prompted intense interest in understanding peptidoglycan biosynthesis in detail. Major advances have been made in recent years with respect to the characterization of key biosynthetic enzymes (3-5). Several structures have been reported for enzymes that catalyze transpeptidation (6, 7), but no PGT structures have been described.PGTs are defined by the presence of five conserved sequence motifs ( Fig. 2A) and exist in two forms: (i) as N-terminal glycosyltransferase domains in bifunctional proteins that also contain a C-terminal transpeptidase domain [called class A penicillin-bindi...

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Glycosyltransferases, Chemistry of

Weadge¹,

Palcic²

2008

Wiley Encyclopedia of Chemical Biology

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Glycosyltransferases (GTs) form glycosidic bonds by catalyzing the transfer of saccharides from a donor to a wide variety of acceptors. The donors used by GTs are sugars conjugated to nucleotides, phosphates, or lipid phosphates; whereas acceptors consist of carbohydrates, proteins, lipids, DNA, and numerous small molecules such as antibiotics, flavonols, steroids, and so on. Together, the products of these reactions comprise the most diverse and abundant class of natural compounds found in nature. Numerous GTs are needed to synthesize these compounds because the formation of each distinct glycosidic linkage requires a different enzyme. The abundance of these enzymes is emphasized by the fact that GTs constitute 1% of the genes in all genomes sequenced. They are ubiquitous in every kingdom of life and in all compartments of the cell. Currently, over 32,000 GT ORFs have been classified into 90 families on the basis of amino acid sequence similarity. The structures for 64 GTs have been determined to date and generally reveal conserved architectures of a GT‐A or GT‐B fold, although other folds have been observed and are predicted. These crystal structures, together with biochemical data, have provided insight into the catalytic mechanism. GTs generally exhibit strict regio/stereospecificity and transfer with either retention or inversion of configuration at the anomeric carbon of the donor sugar. The importance of characterizing the precise activity of these enzymes is exemplified by the many genetic disorders that have been linked to aberrant glycosylation.

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Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases

Cited by 218 publications

References 35 publications

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

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

Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis

Glycosyltransferases, Chemistry of

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