1. Volume 19, Issue 11, Pages 1471-1482 (November 2012)
Active Site Plasticity within the Glycoside Hydrolase NagZ Underlies a Dynamic Mechanism of Substrate Distortion 
John-Paul Bacik, Garrett E. Whitworth, Keith A. Stubbs, David J. Vocadlo, Brian L. Mark 
Chemistry & Biology 
Volume 19, Issue 11, Pages (November 2012)
DOI: /j.chembiol
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2. Figure 1 Catalytic Mechanism of NagZ Enzymes
The enzymes use a two-step, double-displacement mechanism involving the formation and breakdown of a covalent glycosyl-enzyme intermediate via oxocarbenium ion-like transition states (Vocadlo et al., 2000; Vocadlo and Withers, 2005). A histidine is proposed to act as the general acid/base (Litzinger et al., 2010b).
Chemistry & Biology  , DOI: ( /j.chembiol )
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3. Figure 2
Crystal Structures of StNagZ and BsNagZ Bound to Disaccharide Substrate
(A) StNagZ bound to GlcNAc-anhMurNAc (see Figure S1 for synthesis).
(B) BsNagZ bound to GlcNAc-MurNAc (monoclinic form).
(C) BsNagZ bound to distorted GlcNAc-MurNAc (triclinic form).
(D) Superposition of the catalytic domains of BsNagZ and StNagZ.
Crystal structures are shown as cartoon representations with ligands shown as spheres. The conserved NagZ consensus motif [KH(F/I)PG(H/L)GXXXXD(S/T)H] containing the mobile catalytic loop is shown in cyan for StNagZ, green for BsNagZ (monoclinic form), and blue for BsNagZ (triclinic form). Loop thickness is correlated to thermal B factors (thicker loops have higher mobility). The proposed general acid/base histidine is shown in stick format. For BsNagZ, the N-terminal domain is shown in salmon whereas the C-terminal domain is shown in dark salmon. StNagZ and the N-terminal domain of BsNagZ (residues 25–420) adopt a TIM-barrel fold containing the enzyme active site. The C-terminal domain of BsNagZ (residues 421–642) adopts a noncatalytic αβα sandwich fold that does not come into close contact with the enzyme active site. All structural figures were made using PyMOL (DeLano, 2002).
See also Figures S1–S3 and Tables S1 and S2.
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4. Figure 3
Catalytic Loop Mobility and Active Site Architecture of StNagZ and BsNagZ
(A) Superposition of the active site region and catalytic loop (blue) of StNagZ (gray) and BsNagZ (salmon) bound to disaccharide substrate. Only the GlcNAc-MurNAc substrate bound to BsNagZ is shown as sticks for clarity (yellow and cyan carbons are of the substrate bound to the triclinic and monoclinic forms of BsNagZ, respectively; nitrogens are blue, oxygens are red). Dashed lines delimit the distance of the Nε2 atom of the catalytic histidine of each enzyme from the glycosidic oxygen of GlcNAc-MurNAc.
(B) Active site of StNagZ bound to GlcNAc-anhMurNAc (GlcNAc bound as a 4C1 chair).
(C) Model predicting the StNagZ Michaelis complex bound to distorted GlcNAc-anhMurNAc (scissile bond is pseudoaxial). The model was constructed as follows: the anhMurNAc leaving group of GlcNAc-anhMurNAc in the crystallographic StNagZ substrate complex was distorted into a pseudoaxial orientation (which was later verified by superposing this model onto the distorted conformation of GlcNAc-MurNAc in the triclinic BsNagZ complex). A slight rotation of anhMurNAc about the glycosidic bond allowed an effective hydrogen-bonding interaction with Arg70. The conformation of the catalytic loop and position of His176 was modeled based on its conformation in the crystal structure of BsNagZ bound to PUGNAc (Litzinger et al., 2010b). This conformation was also later verified by comparing it to the triclinic BsNagZ substrate complex.
(D) Active site of triclinic BsNagZ bound to distorted GlcNAc-MurNAc (scissile bond is pseudoaxial) with the His234 of the catalytic loop engaging the glycosidic oxygen of the substrate. A small amount of GlcNAc (green carbons) also appears to be present, likely due to slow turnover of the disaccharide. Occupancies for the disaccharide and GlcNAc sugar were refined to 0.66/0.60 and 0.34/0.40, respectively, in both monomers of the asymmetric unit.
(E) Active site of monoclinic BsNagZ bound to GlcNAc-MurNAc (GlcNAc bound as a 4C1 chair). MurNAc lacks interaction with the enzyme, thereby likely allowing greater flexibility of this sugar. For (B)–(E), electron density maps (cyan) are maximum-likelihood weighted 2Fobs − Fcalc syntheses contoured at 1σ around the disaccharide substrates. Hydrogen bonds are shown as yellow dashed lines.
See also Figures S1–S3 and Tables S1 and S2.
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5. Figure 4
Covalent Glycosyl-Enzyme Intermediate and Product Complex of StNagZ
(A) Superposition of unliganded StNagZ (light gray) and StNagZ bound to 5-F-GlcNAc (dark gray).
(B) StNagZ bound to GlcNAc. Active site residues and ligands are drawn as sticks with oxygen and nitrogen atoms shown in red and blue, respectively. Carbon atoms of the enzyme are shown in light or dark gray, whereas the carbon atoms of bound ligands are yellow. Hydrogen bonds are shown as yellow dashed lines. Electron densities (cyan) are maximum-likelihood weighted 2Fobs − Fcalc syntheses contoured at 1σ.
See Tables S1 and S2 as well.
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