Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme’s catalytic mechanism and regulation by GDP-fucose

Somoza, John R.; Menon, Saurabh; Schmidt, Holly; Joseph‐McCarthy, Diane; Dessen, Andréa; Stahl, Mark; Somers, W.S.; Sullivan, Francis X.

doi:10.1016/s0969-2126(00)00088-5

Cited by 90 publications

(116 citation statements)

References 50 publications

(68 reference statements)

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“…Generally, the structure of MUR1 corresponds well with that of the apo E. coli GMD (23), where a slightly better agreement (rmsd of 1.1 Å on CR atoms) occurs with the MUR1 monomer that also lacks bound substrate. The conformations of the small, substrate-binding domains are most variable overall between the two structures and include a large peptide insertion in the E. coli enzyme; however, the position of the three R-helix bundle remains reasonably well conserved.…”

Section: Resultsmentioning

confidence: 62%

“…The extensive interactions between the NADP(H) binding sites observed in the MUR1 tetramer clearly suggest that a dimer-tetramer transition might occur in many GMDs and may be dependent on NADP(H) binding. Interestingly, the apo-form of E. coli GMD was found to be a dimer in the crystal structure and in solution (23). Moreover, recombinant human GMD depleted of NADP(H) was also found to be mainly a dimer (60).…”

Section: Resultsmentioning

confidence: 96%

“…Although L-fucose may be replaced by L-galactose to some extent (51), mur1 plants are characterized by markedly dwarfed growth and compromised tensile stem strength (52). The recent structure of apo-GMD from E. coli (23) established its general tertiary structure, but the absence of crystal structures with bound cofactor or substrate provided little insight into the mechanisms of ligand binding and catalysis. To investigate these issues, we have determined the X-ray crystal structure of the MUR1 enzyme in complexes with the NADP(H) cofactor as well as GDP and GDP-rhamnose, a byproduct formed from the natural MUR1 GDP-4-keto-6-deoxy-D-mannose product during overexpression.…”

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confidence: 99%

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Structure of the MUR1 GDP-Mannose 4,6-Dehydratase from Arabidopsis thaliana: Implications for Ligand Binding and Specificity

et al. 2002

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GDP-D-mannose 4,6-dehydratase catalyzes the first step in the de novo synthesis of GDP-Lfucose, the activated form of L-fucose, which is a component of glycoconjugates in plants known to be important to the development and strength of stem tissues. We have determined the three-dimensional structure of the MUR1 dehydratase isoform from Arabidopsis thaliana complexed with its NADPH cofactor as well as with the ligands GDP and GDP-D-rhamnose. MUR1 is a member of the nucleosidediphosphosugar modifying subclass of the short-chain dehydrogenase/reductase enzyme family, having homologous structures and a conserved catalytic triad of Lys, Tyr, and Ser/Thr residues. MUR1 is the first member of this subfamily to be observed as a tetramer, the interface of which reveals a close and intimate overlap of neighboring NADP + -binding sites. The GDP moiety of the substrate also binds in an unusual syn conformation. The protein-ligand interactions around the hexose moiety of the substrate support the importance of the conserved triad residues and an additional Glu side chain serving as a general base for catalysis. Phe and Arg side chains close to the hexose ring may serve to confer substrate specificity at the O2 position. In the MUR1/GDP-D-rhamnose complex, a single unique monomer within the protein tetramer that has an unoccupied substrate site highlights the conformational changes that accompany substrate binding and may suggest the existence of negative cooperativity in MUR1 function.The 6-deoxy monosaccharide L-fucose is found as a component of glycoconjugates in organisms from bacteria to mammals and has a diverse range of functions. In humans, L-fucose is most notably an important constituent of glycoproteins such as the blood group antigens as well as cell surface carbohydrate ligands of the cell adhesion family of selectins involved in functions such as inflammation and the immune response (1). Among other organisms, L-fucosecontaining glycoconjugates are involved in developmental signaling in Drosophila (2), are critical components of bacterial cell walls where they may play a role in pathogenicity, and among rhizobial organisms, are components of Nod factors, influencing nodulation efficiency and host specificity (3, 4). In plants, L-fucose has important structural functions as a component of glycoproteins and cell wall polysaccharides, such as xyloglucan and rhamnogalacturonans I and II. Xyloglucan molecules cross-link cellulose microfibrils, one of the major load-bearing elements of the cell wall, and may be involved in the regulation of extension growth. It has been proposed that L-fucose may stabilize conformations that effectively bind cellulose (5, 6); however, this hypothesis has recently been challenged on the basis of the normal growth habit and wall strength of an Arabidopsis mutant specifically deficient in xyloglucan fucosylation (7). Although the function of rhamnogalacturonan II is unknown, the presence of fucose appears to be important for formation of the normal borate di-ester cross-linked f...

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Section: Resultsmentioning

confidence: 62%

Section: Resultsmentioning

confidence: 96%

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confidence: 99%

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Structure of the MUR1 GDP-Mannose 4,6-Dehydratase from Arabidopsis thaliana: Implications for Ligand Binding and Specificity

et al. 2002

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“…In this study, Y151F mutation caused an essentially lethal cutback (Ͼ170,000-fold) in catalytic activity of Gdh. Previous replacement of the Tyr equivalent with Phe, however, led to only 190-fold reduction in k cat for RffG (36) and an ϳ2,600-fold decrease in k cat for GMD (40). Our mutagenesis result may suggest that Tyr-151 in Gdh plays a more aggressive role in acting as a general acid/base during chemical catalysis, possibly due to unique characteristics of Gdh protein structure.…”

Section: Discussionmentioning

confidence: 72%

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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“…In bacteria, fucose is found in complex polysaccharides in the cell wall and has been found to be associated with pathogenicity and nodulation (Somoza et al, 2000). The primary biosynthetic route to GDP-fucose is from GDPmannose.…”

Section: Discussionmentioning

confidence: 99%

The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis

et al. 2009

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The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis Azospirillum brasilense is a plant root-colonizing bacterium that exerts beneficial effects on the growth of many agricultural crops. Extracellular polysaccharides of the bacterium play an important role in its interactions with plant roots. The pRhico plasmid of A. brasilense Sp7, also named p90, carries several genes involved in synthesis and export of cell surface polysaccharides. We generated two Sp7 mutants impaired in two pRhico-located genes, noeJ and noeL, encoding mannose-6-phosphate isomerase and GDP-mannose 4,6-dehydratase, respectively. Our results demonstrate that in A. brasilense Sp7, noeJ and noeL are involved in lipopolysaccharide and exopolysaccharide synthesis. noeJ and noeL mutant strains were significantly altered in their outer membrane and cytoplasmic/periplasmic protein profiles relative to the wild-type strain. Moreover, both noeJ and noeL mutations significantly affected the bacterial responses to several stresses and antimicrobial compounds. Disruption of noeL, but not noeJ, affected the ability of the A. brasilense Sp7 to form biofilms. The pleiotropic alterations observed in the mutants could be due, at least partially, to their altered lipopolysaccharides and exopolysaccharides relative to the wild-type.

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Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme’s catalytic mechanism and regulation by GDP-fucose

Cited by 90 publications

References 50 publications

Structure of the MUR1 GDP-Mannose 4,6-Dehydratase from Arabidopsis thaliana: Implications for Ligand Binding and Specificity

Structure of the MUR1 GDP-Mannose 4,6-Dehydratase from Arabidopsis thaliana: Implications for Ligand Binding and Specificity

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

The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis

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