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Literature summary extracted from

  • Lee, C.C.; Ko, T.P.; Chen, C.T.; Chan, Y.T.; Lo, S.Y.; Chang, J.Y.; Chen, Y.W.; Chung, T.F.; Hsieh, H.J.; Hsiao, C.D.; Wang, A.H.
    Crystal structure of PigA a prolyl thioester-oxidizing enzyme in prodigiosin biosynthesis (2019), ChemBioChem, 20, 193-202 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.1.198 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 (Commentary)

EC Number Crystallization (Comment) Organism
2.4.1.198 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

EC Number Protein Variants Comment Organism
2.4.1.198 E244A site-directed mutagenesis Serratia marcescens

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.4.1.198 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

EC Number Organism UniProt Comment Textmining
2.4.1.198 Serratia marcescens Q5W254 i.e. Serratia sp. ATCC39006
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.1.198 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 and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.1.198 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

EC Number Subunits Comment Organism
2.4.1.198 More the enzyme protein folds into a beta-sheet flanked by two alpha-helical domains Serratia marcescens
2.4.1.198 tetramer analytical ultracentrifugation Serratia marcescens

Synonyms

EC Number Synonyms Comment Organism
2.4.1.198 phosphatidylinositol N-acetylglucosaminyltransferase subunit A
-
 Serratia marcescens
2.4.1.198 PigA
-
 Serratia marcescens

Cofactor

EC Number Cofactor Comment Organism Structure
2.4.1.198 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

EC Number General Information Comment Organism
2.4.1.198 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
2.4.1.198 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