Volume 12, Issue 9, Pages 1619-1629 (September 2004) Novel Catalytic Mechanism of Glycoside Hydrolysis Based on the Structure of an NAD+/Mn2+-Dependent Phospho-α-Glucosidase from Bacillus subtilis  Shyamala S. Rajan, Xiaojing Yang, Frank Collart, Vivian L.Y. Yip, Stephen G. Withers, Annabelle Varrot, John Thompson, Gideon J. Davies, Wayne F. Anderson  Structure  Volume 12, Issue 9, Pages 1619-1629 (September 2004) DOI: 10.1016/j.str.2004.06.020

Figure 1 Sequence Alignment of GH4 Family and Comparison with LDH Representative sequences from subsets (α-glucosidases, galactosidases, and β-glucosidases) of GH4 enzymes are aligned: AgaL-Bs, an α-galactosidase from B. subtilis; CelF-Ec, a β-glucosidase from E. coli. Structure-based sequence alignment of LDH (from P. falciparum; 1LDG) and AglA (a GH4 enzyme from T. maritima; 1OBB) with GlvA (from B. subtilis; GlvA-Bs) is also shown. Red asterisks mark residues involved in catalysis and in the coordination of the metal ion. Secondary structural elements shown at the top of the sequences are those of GlvA. The 1° and 2° structures of GlvA are color-coded as follows: N-terminal Rossmann fold region A in blue and the following region B in green. Sequence alignment was done using Multalin/Espript (Gouet et al., 1999; Corpet, 1998) programs and was modified. Structural alignments of GlvA with AglA and LDH were done using algorithms implemented in PdbViewer (Guex and Peitsch, 1997). Structure 2004 12, 1619-1629DOI: (10.1016/j.str.2004.06.020)

Figure 2 Crystal Structure of GlvA (A) A ribbon diagram of the GlvA structure is shown with the N-terminal region in blue, the C-terminal region in pink, and the intervening region in green. Ligands are presented as ball-and-stick with NAD in brick red, Mn2+ in dark pink, and reaction product (G6P) in dark blue. (B) Cα-trace of GlvA tetramer. Within the crystal lattice, a tetramer is formed by the crystallographic 2-fold axes. In this tetramer, interactions across the horizontal 2-fold (x) axis are more extensive than those across the vertical (y) axis. The third (z) axis is perpendicular to the plane of this rendering. The active site pocket is not formed by the intersubunit interfaces, and all the ligands point away from these interfaces. Figures 2 and 3 were prepared in MOLSCRIPT (Kraulis, 1991) and rendered in Raster3D (Merritt and Bacon, 1997). Structure 2004 12, 1619-1629DOI: (10.1016/j.str.2004.06.020)

Figure 3 Structural Alignment of GlvA with LDH (A) Stereo renderings of the Cα traces of GlvA and LDH are presented in dark green and gold, respectively. The two structures were fit by their peptide backbones using algorithms implemented in Swiss-PdbViewer. Ligands are depicted as ball-and-stick models. The GlvA ligands (in darker shades) are NAD in red, Mn2+ in pink, and the G6P in blue; those of LDH are NAD in pale pink and the inhibitor oxamate in cyan. Of particular interest is the orientation and localization of the ligands to the same relative areas in both enzymes. (B and C) Active site architecture of LDH and GlvA. Key interactions between protein and ligands, i.e., the nicotinamide ring of NAD and inhibitor oxamate in LDH (B), and the nicotinamide ring of NAD, reaction product G6P, and Mn2+ in GlvA (C), are presented. (C) also shows the octahedral coordination of Mn2+, which involves interactions with G6P, H2O152, and the conserved Cys171 in a single plane, while the bond with His202 is perpendicular to this plane as is the interaction with the nicotinamide ring of NAD in the opposite direction to that of His202. C atoms are depicted in gray, N in blue, O in red, S in yellow, P in orange, and Mn2+ in pale pink. Important interactions are shown as broken black lines, while some of those directly involved in catalysis are highlighted in green. Structure 2004 12, 1619-1629DOI: (10.1016/j.str.2004.06.020)

Figure 4 Active Site of GlvA (A) Shown here is the Fo − Fc omit map (1.3 σ contour) of the ligand G6P. Also shown are the components believed to be involved in catalysis. This figure was prepared with BOBSCRIPT (Esnouf, 1997) and rendered in Raster3D. (B) Active sites of AglA and GlvA. GlvA and AglA (1OBB [Lodge et al., 2003]) were aligned in Swiss PdbViewer via their main chain atoms, and the superposition of their active site residues is shown here. The ligands, NAD(H), metal, and G6P of GlvA are shown in pale pink, while the maltose substrate and NAD(H) of AglA are presented in maroon. The protein residues belonging to GlvA are shown in aquamarine, while those of AglA are shown in gray with O, N, and S atoms colored red, blue, and yellow, respectively. Of interest is the nonsuperposition of the ligands of AglA on to those of GlvA, while the protein residues align reasonably well. Structure 2004 12, 1619-1629DOI: (10.1016/j.str.2004.06.020)

Figure 5 Proposed Mechanism of Glucoside Hydrolysis by GlvA Structure 2004 12, 1619-1629DOI: (10.1016/j.str.2004.06.020)