Volume 12, Issue 6, Pages 927-935 (June 2004) Structure and Mechanism of GDP-Mannose Glycosyl Hydrolase, a Nudix Enzyme that Cleaves at Carbon Instead of Phosphorus  Sandra B Gabelli, Mario A Bianchet, Hugo F Azurmendi, Zuyong Xia, Vibhor Sarawat, Albert S Mildvan, L.Mario Amzel  Structure  Volume 12, Issue 6, Pages 927-935 (June 2004) DOI: 10.1016/j.str.2004.03.028

Figure 1 Sequence Alignment of Homologous GDPMH in Enterobacteria and Vibrio Secondary structure elements of E. coli GDPMH are shown as coils (helices) and arrows (strands). Sequence similarities are boxed with white background, and sequence identities are boxed with pink background. The modified Nudix sequence runs from residue 51 to 73 and is shown in green. Residues involved in GDP recognition are marked with asterisks, and residues at hydrogen bonding distance to a bound Tris molecule are marked with black triangles. Residues involved in dimer interface or crystal contacts are marked with “B.” Alignment was prepared with Clustal X and ESPrit. Structure 2004 12, 927-935DOI: (10.1016/j.str.2004.03.028)

Figure 2 Ribbon Diagram of the Structure of the GDPMH in Complex with Mg2+-GDP One monomer is shown in gray and the other in yellow. The Mg2+ atom is shown in green. Ligands of the Mg2+ ion (Gly50, Glu70, and Gln123) as well as GDP are shown as stick models. Structure 2004 12, 927-935DOI: (10.1016/j.str.2004.03.028)

Figure 3 Stereo View of the Electron Density of GDP Bound to GDP-Mannose Hydrolase The 2Fo-Fc electron density map (blue) was contoured at a 1σ level. Residues involved in substrate recognition are shown with colored bonds. Structure 2004 12, 927-935DOI: (10.1016/j.str.2004.03.028)

Figure 4 Binding of Tris and GDP-Mannose to GDPMH (A) Residues involved in binding a Tris molecule present in the crystal structure are shown in the ball-and-stick representation. Hydrogen bonds are represented as dashed lines. (B) Binding of mannose to GDPMH was modeled as described in Experimental Procedures. The modeled mannose is covalently bound to one of the oxygen atoms of the β-phosphate of GDP. (The distance between O3 of mannose and the Nδ of His88 is 3.8 Å, but the hydrogen bond is shown because a small rearrangement of the histidine side chain can result in a shorter distance.) Structure 2004 12, 927-935DOI: (10.1016/j.str.2004.03.028)

Figure 5 Coordination Geometry of the Mg2+ Bound in the GDPMH-Mg2+-GDP Complex Structure 2004 12, 927-935DOI: (10.1016/j.str.2004.03.028)

Figure 6 Schematic Diagram of the Substrate and Residues Involved in Catalysis and Alternative Mechanisms of GDPMH (A) Schematic diagram of the substrate and residues involved in catalysis. The numbers in parentheses show the factors by which kcat is decreased in mutating these residues. (B and C) Alternative mechanisms of GDPMH. These mechanisms are consistent with the 1.3 Å X-ray structure (see text) and with the effects of mutation on catalysis reported here and previously. Structure 2004 12, 927-935DOI: (10.1016/j.str.2004.03.028)

Figure 7 Nudix Structural Motif in ADPRase and GDPMH (A) ADPRase. Side chains of residues that form part of the Nudix signature sequence are shown in an all-atom representation. ADP-ribose and the metal ions (in green) are also shown. (B) Equivalent representation of the residues of the GDPMH Nudix motif. GDP and the Mg2+ ion are also shown. Structure 2004 12, 927-935DOI: (10.1016/j.str.2004.03.028)

Figure 8 Sequence and Structure Alignments of ADPRase and GDPMH Showing the Effects of the Six-Residue Shortening of Loop 9 The main chain of ADPRase is shown in green and that of GDPMH in copper. The positions of the two catalytic bases, Glu162 (ADPRase) and His124 (GDPMH), are shown as are the structures of the two substrates. The candidates for entering water molecules in the substitution mechanism are also shown. Structure 2004 12, 927-935DOI: (10.1016/j.str.2004.03.028)