Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa.
The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-alpha-ketobutyrate. It belongs to the D-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B6 (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 A from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 A. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (VM) of 3.64 A3 Da(-1) and a solvent content of 66%.
Ha
Jun Yong
JY
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, South Korea.
Lee
Ji Hyun
JH
Kim
Kyoung Hoon
KH
Kim
Do Jin
DJ
Lee
Hyung Ho
HH
Kim
Hye-Kyung
HK
Yoon
Hye-Jin
HJ
Suh
Se Won
SW
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D002237:Carbohydrate Dehydrogenases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D000429:Alcohol Oxidoreductases; D002237:Carbohydrate Dehydrogenases; C083779:2-hydroxyacid dehydrogenase; C061651:pdxB protein, E coli