Cloned (Comment) | Organism |
---|---|
gene pigA, recombinant expression of codon-optimmized gene encoding the C-terminally His6-tagged wild-type and mutant enzymes with TEV protease cleavage site in Escherichia coli strain BL21(DE3) | Serratia marcescens |
Crystallization (Comment) | Organism |
---|---|
purified recombinant PigA in apoform and in complex with cofactor FAD or L-proline, sitting drop vapor diffusion method, mixing of 0.001 ml of 15 mg/ml protein in 100 mm NaCl and 50 mm Tris·HCl, pH 8.0, with 0.001 ml of reservoir solution containing 27% PEG 600, and 0.1 M Na-HEPES, pH 7.5, for the complex crystals FAD is added in a molar ratio of 3:1 ratio to the protein solution, and mixed with a reservoir solution containing 40% ethylene glycol and 0.1 M Na-acetate, pH 5.0; or 1.7 M (NH4)2SO4, 0.2 M NaCl, and 0.1 M sodium cacodylate, pH 6.5, for the proline-complex crystal the protein solution contains 7 mM L-proline in addition to FAD and crown ether, and the reservoir contains 1.9 M sodium malonate, pH 6.0. The Pro-Gly complex crystal is obtained by including 5 mM L-prolylglycine in the E244A protein solution, with a reservoir of 1.6 M (NH4)2SO4, 2% PEG 400, and 0.1 M citric acid, pH 4.0, X-ray diffraction structure determination and analysis at 1.3-2.55 A resolution, molecular replacement using the structure of butyryl-CoA dehydrogenase (PDB ID 4L1F) as a search model | Serratia marcescens |
Protein Variants | Comment | Organism |
---|---|---|
E244A | site-directed mutagenesis | Serratia marcescens |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
UDP-N-acetyl-alpha-D-glucosamine + 1-phosphatidyl-1D-myo-inositol | Serratia marcescens | - |
UDP + 6-(N-acetyl-alpha-D-glucosaminyl)-1-phosphatidyl-1D-myo-inositol | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Serratia marcescens | Q5W254 | i.e. Serratia sp. ATCC39006 | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage by TEV protease, and ultrafiltration | Serratia marcescens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
UDP-N-acetyl-alpha-D-glucosamine + 1-phosphatidyl-1D-myo-inositol | - |
Serratia marcescens | UDP + 6-(N-acetyl-alpha-D-glucosaminyl)-1-phosphatidyl-1D-myo-inositol | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | the enzyme protein folds into a beta-sheet flanked by two alpha-helical domains | Serratia marcescens |
tetramer | analytical ultracentrifugation | Serratia marcescens |
Synonyms | Comment | Organism |
---|---|---|
phosphatidylinositol N-acetylglucosaminyltransferase subunit A | - |
Serratia marcescens |
PigA | - |
Serratia marcescens |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | enzyme binding structure analysis, overview. The variable conformations of loop beta4-beta5 and helix alphaG correlate well with the structural flexibility required for substrate entrance to the Re side of FAD | Serratia marcescens |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the acyl coenzyme A dehydrogenase (ACAD) family member, thus the enzyme protein folds into a beta-sheet flanked by two alpha-helical domains | Serratia marcescens |
additional information | modeling with PigG, the acyl carrier protein, suggests a reasonable mode of interaction with PigA. The structure helps to explain the proline oxidation mechanism, in which Glu244 plays a central role by abstracting the substrate protons. It also reveals a plausible pocket for oxygen binding to the Si side of FAD | Serratia marcescens |