2.3.1.157: glucosamine-1-phosphate N-acetyltransferase

288687, 288688

hanging drop vapor diffusion method. X-ray crystal structure of the bifunctional enzyme in complex with UDP-GlcNAc and CoA, determined to 2.1 A resolution and reveals a two-domain architecture that is responsible for the two reactions of EC 2.3.1.157 and EC 2.7.7.23. The C-terminal domain is responsible for the CoA-dependent acetylation of Glc-1-phosphate to GlcNAc-1-phosphate and displays the longest left-handed parallel alpha-helix observed to date. The acetyltransferase active site defined by the binding site for CoA makes use of residues from all three subunits and is positioned beneath an open cavity large enough to accommodate the Glc-1-PO4 acetyl acceptor. The N-terminal domain catalyzes uridyl transfer from UTP to GlcNAc-1-phosphate to form the final products UDP-GlcNAc and pyrophosphate

P0ACC7

643075

in complex with acetyl-CoA, CoA and glucosamine-1-phosphate, and with desulfo-CoA and N-acetylglucosamine-1-phosphate. The 2-amino group of glucosamine-1-phosphate is positioned in proximity to the acetyl-CoA to facilitate direct attack on its thioester by a ternary complex mechanism

677026

purified GlmU, hanging drop vapour diffusion method, mixing of 8 mg/ml enzyme in 50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 2 mM TCEP, with 2 mM inhibitor ligand, and with reservoir solution containing 1922% w/v PEG 3350 and either 100 mM PCTP, pH 5.0, 100-200 mM ammonium sulfate or 100 mM MMT, pH 6.0-7.0, and 400 mM ammonium sulfate, 20°C, 2-7 days, X-ray diffraction structure determination and analysis

719064

apo GlmU is crystallized at 20 °C using the sitting-drop vapor diffusion methodcrystal structure of a mimic of the Michaelis complex, with glucose 1-phosphate and acetyl-coenzyme A, helps us to propose the residues involved in deprotonation of glucosamine 1-phosphate and the loop movement that likely generates the active site required for glucosamine 1-phosphate to bind

A5U161

756049

apo GlmU is crystallized at 20 °C using the sitting-drop vapor diffusion methodcrystal structure of a mimic of the Michaelis complex, with glucose 1-phosphate and acetyl-coenzyme A, indicates the residues involved in deprotonation of glucosamine 1-phosphate and the loop movement that likely generates the active site required for glucosamine 1-phosphate to bind

A5U161

756049

crystal structures of GlmU in apo form and UDP-N-acetylglucosamine-bound form is determined. The structure shows a two-domain architecture, with an N-terminal domain having an alpha/beta-like fold and with a C-terminal domain that forms a left-handed parallel beta-helix structure

705135

crystallized using the hanging drop vapour-diffusion method. Native diffraction data are collected from crystals belonging to space group R32 and processed to a resolution of 2.2 A

706622

docking surface representation of the GlmU allosteric site in complex with inhibitor (4Z)-4-(4-benzyloxybenzylidene)-2-(naphthalen-2-yl)-1,3-oxazol-5(4H)-one. Residues Tyr150, Glu250 and Arg 253 are in hydrogen bonding with carbonyl oxygen over the oxazole ring. Leu144, Pro147, Phe148, Tyr150, Ala233, Ala236 and Leu247 participate in strong hydrophobic interactions

737150

GlmUMtb in complex with substrates/products bound at the acetyltransferase active site, sitting drop vapor diffusion method, mixing of 400 nl of 15 mg/ml protein, 5 mM acetyl-CoA, 5 mM MgCl2, 5 mM UDP-GlcNAc with 400 nl of 18% PEG 3350, 0.1 M Tris-Cl, pH 8.5, and 2% tacsimate, 4-8 days, for enzyme complex with CoA and N-acetylglucosamine-1-phosphate, acetyl-Coa-containing crystals are soaked in 5 mM GlcN-1-P, 5 mM MgCl2, 5 mM UDP-GlcNAc, 5 mM acetyl-CoA, 18% PEG 3350, 0.1 M Tris-Cl, pH 8.5, and 2% tacsimate, or by co-crystallizing the enzyme with 5 mM GlcNAc-1-P, 5 mM MgCl2, 5 mM UDPGlcNAc, and 5 mM CoA under the conditions mentioned for obtaining GlmUMtb(AcCoA) crystals, X-ray diffraction structure determination and analysis at 1.98-2.33 A resolution

720042

in complex with acetyl-CoA and glucose 1-phosphate

A5U161

756049

purified GlmU, sitting drop vapor diffusion method, mixing of 400 nl of 15 mg/ml GlmU in 5 mM acetyl-CoA, 5 mM MgCl2, 5 mM UDP-GlcNAc with 400 nL of 18% PEG 3350, 0.1 M Tris-Cl, pH 8.5, and 2% tacsimate, 7-8 days, for coupling to acetyl-CoA, crystals are soaked in 5 mM GlcN-1-P, 5 mM MgCl2, 5 mM UDP-GlcNAc, 5 mM acetyl-CoA, 18% PEG 3350, 0.1 M Tris-Cl, pH 8.5, and 2% tacsimate, or in 5 mM GlcNAc-1-P, 5 mM MgCl2, 5 mM UDP-GlcNAc, 5 mM CoA, X-ray diffraction structure determination and analysis at 1.98-2.33 A resolution

720042

structure of enzyme bound to ATP and N-acetyl-D-glucosamine 1-phosphate

735396

the crystal structures of MtGlmU in an unliganded form and in complexes in which either GlcNAc-1-P or UDP-GlcNAc occupies the uridyltransferase active site are determined using the sitting-drop vapour-diffusion method. These structures identify the active-site contacts between protein and ligands and suggest a ternary-complex mechanism of action for the GlmU uridyltransferase reaction

701468

using the hanging-drop vapour-diffusion method. GlmU also crystallizes in the cubic space group I432. It diffracts to 3.4 A resolution. This poor diffraction is correlated with a sparse crystal packing leading to the presence of large solvent channels in the crystal, unlike the hexagonal forms. The distinct crystal packing in these two forms may be a result of the involvement of different surfaces in crystal contacts