Journal: Biochemistry

Article Title: Structural and Functional Studies of WlbA: a Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-Dideoxy-D-Mannuronic Acid

doi: 10.1021/bi101103s

Figure Lengend Snippet: Double reciprocal plots of initial rates of formation of UDP-3-keto-GlcNAcA using the enzyme from T. thermophilus. The concentration of α-ketoglutarate was varied at 10.0, 3.3, 2.0, and 1.0 mM at several fixed concentrations of UDP-GlcNAcA: 2.0 mM (diamond), 0.3 mM (square), 0.15 mM (triangle), and 0.1 mM (circle). Initial velocities (v) are expressed in micromolar/min of UDP-3-keto-GlcNAcA.

Article Snippet: In order to produce diffraction quality crystals of the appropriate enzyme/ligand complexes required for this investigation, two different versions of WlbA were cloned and purified, namely one from P. aeruginosa PAO1-LAC (ATCC 47085) and the other from Thermus thermophilus (ATCC BAA-163).

Techniques: Concentration Assay

Journal: Biochemistry

Article Title: Structural and Functional Studies of WlbA: a Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-Dideoxy-D-Mannuronic Acid

doi: 10.1021/bi101103s

Figure Lengend Snippet: The structure of the WlbA/NAD(H)/α-ketoglutarate complex from T. thermophilus. Electron densities corresponding to the bound NAD(H) and α-ketoglutarate ligands in Chain B are presented in (a). The electron density map was calculated as described in figure legend 2 and was contoured at 2.5 σ. Those residues lying within 3.2 Å of the α-ketoglutarate are shown in (b). Possible hydrogen bonding interactions are indicated by the dashed lines, and ordered water molecules are depicted as red spheres. With the exception of Lys 101, all other residues involved in the binding of α-ketoglutarate are contributed by the C-domain, which is highlighted in green. The red dashed line indicates a distance of 3.1 Å between the C-2 carbon of α-ketoglutarate and the nicotinamide C-4 carbon.

Article Snippet: In order to produce diffraction quality crystals of the appropriate enzyme/ligand complexes required for this investigation, two different versions of WlbA were cloned and purified, namely one from P. aeruginosa PAO1-LAC (ATCC 47085) and the other from Thermus thermophilus (ATCC BAA-163).

Techniques: Binding Assay

Journal: Biochemistry

Article Title: Structural and Functional Studies of WlbA: a Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-Dideoxy-D-Mannuronic Acid

doi: 10.1021/bi101103s

Figure Lengend Snippet: The structure of the WlbA/NAD(H)/UDP-GlcNAcA complex from T. thermophilus. Electron densities corresponding to the bound NAD(H) and UDP-GlcNAcA ligands in Chain C are presented in (a). The electron density map was calculated as described in figure legend 2 and was contoured at 2.5 σ. Those residues lying within 3.2 Å of UDP-GlcNAcA are shown in (b). Possible hydrogen bonding interactions are indicated by the dashed lines, and ordered water molecules are depicted as red spheres. With the exception of Tyr 12 and Lys 101, all other residues involved in the binding of the UDP-linked sugar are contributed by the C-domain, which is highlighted in green. The red dashed line indicates a distance of 3.0 Å between the C-3′ carbon of the hexose and the nicotinamide C-4 carbon.

Article Snippet: In order to produce diffraction quality crystals of the appropriate enzyme/ligand complexes required for this investigation, two different versions of WlbA were cloned and purified, namely one from P. aeruginosa PAO1-LAC (ATCC 47085) and the other from Thermus thermophilus (ATCC BAA-163).

Techniques: Binding Assay

Journal: Biochemistry

Article Title: Structural and Functional Studies of WlbA: a Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-Dideoxy-D-Mannuronic Acid

doi: 10.1021/bi101103s

Figure Lengend Snippet: Comparison of the T. thermus WlbA and Z. mobilis GFOR structures. A ribbon representation of the GFOR tetramer is presented in (a). Note the stunning difference between its quaternary structure compared to that of WlbA shown in Figure 2a. A superposition of a subunit of GFOR, displayed in purple, onto a subunit of WlbA, colored in light blue, is presented in (b). The positions of the bound ligands are displayed in stick representations. These include NAD(H) and α-ketoglutarate in WlbA (gray bonds) and NADP(H) and glycerol (gold bonds) in GFOR. There are three major conformational differences between these two enzymes that account for their widely differing quaternary structures as indicated by Regions 1, 2, and 3. X-ray coordinates for GFOR were obtained from the Protein Data Bank (accession no. 1H6B).

Article Snippet: In order to produce diffraction quality crystals of the appropriate enzyme/ligand complexes required for this investigation, two different versions of WlbA were cloned and purified, namely one from P. aeruginosa PAO1-LAC (ATCC 47085) and the other from Thermus thermophilus (ATCC BAA-163).

Techniques: