gene wecD, expression of N-terminal fusion protein with a noncleavable His6-tag in Escherichia coli strain BL21(DE3)

Escherichia coli

purified recombinant N-terminally His6-tagged WecD free and in complex with acetyl-CoA, hanging drop vapour diffusion method, mixing of 0.001 ml of 7.1 mg/ml protein in a 20 mM Tris-Cl, pH 8.0, 450 mM NaCl, and 5 mM DTT, with 0.001 ml reservoir solution aontaining 100 mM Na-acetate, pH 4.5, 10 mM ZnSO4, 20°C, 1 day, X-ray diffraction structure determination and analysis at 1.66-1.95 A resolution

Escherichia coli

acetyl-CoA + dTDP-4-amino-4,6-dideoxy-alpha-D-galactose

Escherichia coli

CoA + dTDP-4-acetamido-4,6-dideoxy-alpha-D-galactose

?

acetyl-CoA + dTDP-4-amino-4,6-dideoxy-alpha-D-galactose

Escherichia coli CFT073

CoA + dTDP-4-acetamido-4,6-dideoxy-alpha-D-galactose

?

Escherichia coli

Q8FBQ3

gene wecD

Escherichia coli CFT073

Q8FBQ3

gene wecD

recombinant N-terminally His6-tagged WecD from Escherichia coli strain BL21(DE3)

Escherichia coli

acetyl-CoA + dTDP-4-amino-4,6-dideoxy-alpha-D-galactose

719706

Escherichia coli

CoA + dTDP-4-acetamido-4,6-dideoxy-alpha-D-galactose

?

acetyl-CoA + dTDP-4-amino-4,6-dideoxy-alpha-D-galactose

acetyl-CoA binding site structure, overview

719706

Escherichia coli

CoA + dTDP-4-acetamido-4,6-dideoxy-alpha-D-galactose

?

acetyl-CoA + dTDP-4-amino-4,6-dideoxy-alpha-D-galactose

719706

Escherichia coli CFT073

CoA + dTDP-4-acetamido-4,6-dideoxy-alpha-D-galactose

?

acetyl-CoA + dTDP-4-amino-4,6-dideoxy-alpha-D-galactose

acetyl-CoA binding site structure, overview

719706

Escherichia coli CFT073

CoA + dTDP-4-acetamido-4,6-dideoxy-alpha-D-galactose

?

dimer

WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, gel filtration and dynamic light scattering, three-dimensional structure comparison, overview

Escherichia coli

TDP-fucosamine acetyltransferase

Escherichia coli

WecD

Escherichia coli

acetyl-CoA

Escherichia coli

gene wecD, expression of N-terminal fusion protein with a noncleavable His6-tag in Escherichia coli strain BL21(DE3)

Escherichia coli

acetyl-CoA

Escherichia coli

purified recombinant N-terminally His6-tagged WecD free and in complex with acetyl-CoA, hanging drop vapour diffusion method, mixing of 0.001 ml of 7.1 mg/ml protein in a 20 mM Tris-Cl, pH 8.0, 450 mM NaCl, and 5 mM DTT, with 0.001 ml reservoir solution aontaining 100 mM Na-acetate, pH 4.5, 10 mM ZnSO4, 20°C, 1 day, X-ray diffraction structure determination and analysis at 1.66-1.95 A resolution

Escherichia coli

acetyl-CoA + dTDP-4-amino-4,6-dideoxy-alpha-D-galactose

Escherichia coli

CoA + dTDP-4-acetamido-4,6-dideoxy-alpha-D-galactose

?

acetyl-CoA + dTDP-4-amino-4,6-dideoxy-alpha-D-galactose

Escherichia coli CFT073

CoA + dTDP-4-acetamido-4,6-dideoxy-alpha-D-galactose

?

recombinant N-terminally His6-tagged WecD from Escherichia coli strain BL21(DE3)

Escherichia coli

acetyl-CoA + dTDP-4-amino-4,6-dideoxy-alpha-D-galactose

719706

Escherichia coli

CoA + dTDP-4-acetamido-4,6-dideoxy-alpha-D-galactose

?

acetyl-CoA + dTDP-4-amino-4,6-dideoxy-alpha-D-galactose

acetyl-CoA binding site structure, overview

719706

Escherichia coli

CoA + dTDP-4-acetamido-4,6-dideoxy-alpha-D-galactose

?

acetyl-CoA + dTDP-4-amino-4,6-dideoxy-alpha-D-galactose

719706

Escherichia coli CFT073

CoA + dTDP-4-acetamido-4,6-dideoxy-alpha-D-galactose

?

acetyl-CoA + dTDP-4-amino-4,6-dideoxy-alpha-D-galactose

acetyl-CoA binding site structure, overview

719706

Escherichia coli CFT073

CoA + dTDP-4-acetamido-4,6-dideoxy-alpha-D-galactose

?

dimer

WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, gel filtration and dynamic light scattering, three-dimensional structure comparison, overview

Escherichia coli

physiological function

the enzyme is involved in biosynthesis of enterobacterial common antigen, ECA, a polysaccharide found on the outer membrane of virtually all Gram-negative enteric bacteria, that consists of three sugars, N-acetyl-D-glucosamine, N-acetyl-D-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-D-galactose, organized into trisaccharide repeating units having the sequence ->3)-alpha-D-Fuc4NAc-(1->4)-beta-D-ManNAcA-(1->4)-alpha-D-GlcNAc-(1->

Escherichia coli

evolution

WecD is a GNAT family member. The GNAT N-acetyltransferase fold is common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars

Escherichia coli

usage of flexible docking to generate a WecD-bound model of the acetyl-CoATDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of CoA, modeling of TDP-D-Fuc4N:acetyl-CoA transition state bound to WecD, overview. The WecD tetrahedral intermediate results from nucleophilic attack of the 4-amino group of TDP-fucosamine at the acetyl-CoA thioester carbon atom

Escherichia coli

physiological function

the enzyme is involved in biosynthesis of enterobacterial common antigen, ECA, a polysaccharide found on the outer membrane of virtually all Gram-negative enteric bacteria, that consists of three sugars, N-acetyl-D-glucosamine, N-acetyl-D-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-D-galactose, organized into trisaccharide repeating units having the sequence ->3)-alpha-D-Fuc4NAc-(1->4)-beta-D-ManNAcA-(1->4)-alpha-D-GlcNAc-(1->

Escherichia coli

evolution

WecD is a GNAT family member. The GNAT N-acetyltransferase fold is common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars

Escherichia coli

usage of flexible docking to generate a WecD-bound model of the acetyl-CoATDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of CoA, modeling of TDP-D-Fuc4N:acetyl-CoA transition state bound to WecD, overview. The WecD tetrahedral intermediate results from nucleophilic attack of the 4-amino group of TDP-fucosamine at the acetyl-CoA thioester carbon atom

Escherichia coli