reaction energetics and mechanism, active site structure, substrate binding structure, determination of conformation states of the enzyme during reaction from apo to single occupied transitional binding state and to fully occupied state
In Escherichia coli, the coenzyme pyridoxal 5'-phosphate is synthesized de novo by a pathway that involves EC 188.8.131.52 (erythrose-4-phosphate dehydrogenase), EC 184.108.40.2060 (4-phosphoerythronate dehydrogenase), EC 220.127.116.11 (phosphoserine transaminase), EC 18.104.22.1682 (4-hydroxythreonine-4-phosphate dehydrogenase), EC 22.214.171.124 (pyridoxine 5'-phosphate synthase) and EC 126.96.36.199 (with pyridoxine 5'-phosphate as substrate). 1-Deoxy-D-xylulose cannot replace 1-deoxy-D-xylulose 5-phosphate as a substrate .
the enzyme shows phosphomutase activity transfering the 5-phosphoryl group of 1-deoxy-D-xylose 5-phosphate to the 4-hydroxyl at some point in the reaction, the phosphate group of the substrate is required for activity, reaction mechanism, overview
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12 mg/ml purified recombinant enzyme, wild-type or selenomethionine-labeled, in 10 mM Tris-HCl, pH 7.5, 0.4 mM potassium phosphate, 1.21 mM NaCl, 8% PEG 8000, 9% PEG 1000, 10% glycerol, and 4 mM 1-deoxy-D-xylulose, hanging drop vapour diffusion method, X-ray diffraction structure determination and analysis at 1.96-3.2 A resolution
6 mg/ml purified recombinant enzyme in 2 mM Tris-HCl, pH 8.0, 0.003 ml mixed with 0.0015 ml precipitation solution, equilibration against 0.5 ml reservoir solution, sitting drop vapour diffusion method, method 1: 0.1 M sodium acetate, pH 4.6, 8% PEG 4000 as precipitant and 0.1 M L-cysteine as additive, triangular-shaped crystals within 10 days, method 2: precipitant solution contains 10% PEG 6000, 2 M NaCl, slow crystal growth, 6 weeks, microseeding into protein solution of 13.5 mg/ml protein, 2 mM Tris-HCl, pH 8.8, 1 day, or hanging drop vapour diffusion method over 1 week, method evaluation, X-ray diffraction and preliminary structure determination and analysis at 2.6 A resolution
purified enzyme in complex with products pyridoxine 5'-phosphate and phosphate, the enzyme crystals are incubated with 5 mM product solution for 1 h, X-ray diffraction structure determination and analysis at 2.3 A resolution
purified enzyme, the native enzyme crystals are incubated with 2.5 mM glyceraldehyde 3-phosphate and 2.5 mM 1-deoxy-D-xylulose 5-phosphate for 3 hours, in 10 mM or 100 mM phosphate for 3 days, and for another 3 days in 20 mM 1-deoxy-D-xylulose 5-phosphate, X-ray diffraction structure determination and analysis at 2.3 A resolution