Toward a Structural Understanding of the Dehydratase Mechanism

Allard, S.T.M.; Beis, Konstantinos; Giraud, Marie‐France; Hegeman, Adrian D.; Gross, Janet L.; Wilmouth, R.C.; Whitfield, Chris; Graninger, Michael; Messner, Paul; Allen, Andrew G.; Maskell, Duncan J.; Naismith, James H.

doi:10.1016/s0969-2126(01)00694-3

Cited by 96 publications

(149 citation statements)

References 61 publications

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“…The Ser/Thr side chain may play a role in the proper orientation of bound substrate and/or in the stabilization of reaction intermediates or transition states. Unusually short (<2.5 Å) hydrogen bonds between this side chain and the hexose O4 hydroxyl were noted for the productive ternary complex in SQD1 (22) and the abortive complexes of GalE (28) and Rm1B (31). The presence of short, low-barrier hydrogen bonds at this position has been proposed to facilitate proton transfer during catalysis (21,22,28).…”

Section: Resultsmentioning

confidence: 99%

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: 99%

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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“…8, steps I3 IV3 V). The final outcome of the C6 dehydratase reaction produces a C4 ketone (30), although the diol forms are depicted in Fig. 8 as the keto group rapidly converts to a diol in aqueous solution.…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of Dehydratase/Aminotransferase Pairs from Helicobacter and Campylobacter

Schoenhofen

McNally

Vinogradov

et al. 2006

Journal of Biological Chemistry

153

188

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“…The presence of a tightly bound NADP ϩ is essential for the proposed catalytic activity of GMD (34,35), as demonstrated by the complete loss of activity by the apoenzyme (14). However, recent GMD crystal structures at 2.2 Å resolution from A. thaliana and P. aeruginosa indicate unambiguously the presence of NADPH, and not NADP ϩ , bound to the proteins (15)(16).…”

Section: Discussionmentioning

confidence: 99%

Differential Role of NADP+ and NADPH in the Activity and Structure of GDP-D-mannose 4,6-Dehydratase from Two Chlorella Viruses

Fruscione¹,

Sturla²,

Duncan

et al. 2008

Journal of Biological Chemistry

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GDP-D-mannose 4,6-dehydratase (GMD) is a key enzyme involved in the synthesis of 6-deoxyhexoses in prokaryotes and eukaryotes. Paramecium bursaria chlorella virus-1 (PBCV-1) encodes a functional GMD, which is unique among characterized GMDs because it also has a strong stereospecific NADPHdependent reductase activity leading to GDP-D-rhamnose formation (Tonetti, M., Zanardi, D., Gurnon, J., Fruscione, F., Armirotti, A., Damonte, G., Sturla, L., De Flora, A., and Van Etten, J.L. (2003) J. Biol. Chem. 278, 21559 -21565). In the present study we characterized a recombinant GMD encoded by another chlorella virus, Acanthocystis turfacea chlorella virus 1 (ATCV-1), demonstrating that it has the expected dehydratase activity. However, it also displayed significant differences when compared with PBCV-1 GMD. In particular, ATCV-1 GMD lacks the reductase activity present in the PBCV-1 enzyme. Using recombinant PBCV-1 and ATCV-1 GMDs, we determined that the enzymatically active proteins contain tightly bound NADPH and that NADPH is essential for maintaining the oligomerization status as well as for the stabilization and function of both enzymes. Phylogenetic analysis indicates that PBCV-1 GMD is the most evolutionary diverged of the GMDs. We conclude that this high degree of divergence was the result of the selection pressures that led to the acquisition of new reductase activity to synthesize GDP-D-rhamnose while maintaining the dehydratase activity in order to continue to synthesize GDP-L-fucose.GDP-D-mannose 4,6-dehydratase (GMD, 2 EC 4.2.1.47) is the first enzyme in the biosynthetic pathways that leads to the formation of several deoxyhexoses, such as GDP-L-fucose (1), GDP-D-rhamnose (2), GDP-6-deoxy-D-talose (3), and GDP-Dperosamine (4). Genes encoding GMDs have been identified in most prokaryotes and eukaryotes (5-8). Recently, we characterized the first GMD encoded by a virus, Paramecium bursaria chlorella virus 1 (PBCV-1) (9). PBCV-1 is the prototype of a group (family Phycodnaviridae) of large, icosahedral, plaqueforming, double-stranded DNA viruses that replicate in certain unicellular, eukaryotic chlorella-like green algae (10). In nature, the chlorella host is a hereditary endosymbiont in the ciliated protozoan P. bursaria. PBCV-1 has ϳ365 protein-encoding genes, including genes that encode several proteins that resemble enzymes involved in the metabolism and processing of sugars; these proteins include five putative glycosyltransferases believed to be involved in the glycosylation of the virus major capsid protein (11). PBCV-1 also encodes the two enzymes involved in GDP-Lfucose biosynthesis, GMD and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GMER) (Fig. 1) (9).GMD is a member of the short-chain dehydrogenase/reductase family of proteins (12) and in general has structural features compatible with enzymes belonging to the reductase-epimerase-dehydrogenase superfamily (13). GMD sequences are well conserved among organisms; they all contain three conserved amino acids, Ser/Thr, Tyr, and Lys, involved in ca...

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Toward a Structural Understanding of the Dehydratase Mechanism

Cited by 96 publications

References 61 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 of Dehydratase/Aminotransferase Pairs from Helicobacter and Campylobacter

Differential Role of NADP+ and NADPH in the Activity and Structure of GDP-D-mannose 4,6-Dehydratase from Two Chlorella Viruses

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