Cloning, expression, purification and crystallization of dihydroxybutanone phosphate synthase from Magnaporthe grisea.

Dihydroxybutanone phosphate synthase (DS) catalyzes a commitment step in riboflavin biosynthesis where ribulose 5-phosphate is converted to dihydroxybutanone phosphate and formate. DS was cloned from the pathogenic fungus Magnaporthe grisea (using functional complementation of an Escherichia coli DS knockout mutant) and expressed in E. coli. The purified protein crystallized in space group P2(1)2(1)2. Diffraction data extending to 1.5, 1.0 and 1.8 A resolution were collected from crystals that were divalent cation free, soaked in Zn(2+) or soaked in Mg(2+), respectively.

[1]  A. Bacher,et al.  Biosynthesis of riboflavin. Studies on the mechanism of L-3,4-dihydroxy-2-butanone 4-phosphate synthase. , 1991, The Journal of biological chemistry.

[2]  A. Bacher,et al.  Biosynthesis of riboflavin: cloning, sequencing, and expression of the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli , 1992, Journal of bacteriology.

[3]  G. Lorimer,et al.  GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli , 1989, Nature.

[4]  A. Bacher,et al.  NMR studies on the 46-kDa dimeric protein, 3,4-dihydroxy-2-butanone 4-phosphate synthase, using 2H, 13C, and 15N-labelling. , 1999, European journal of biochemistry.

[5]  D. Jordan,et al.  Rate Limitations in the Lumazine Synthase Mechanism , 2000 .

[6]  D. Jordan,et al.  Plant Riboflavin Biosynthesis , 1999, The Journal of Biological Chemistry.

[7]  A. Bacher,et al.  Studies on the 4-carbon precursor in the biosynthesis of riboflavin. Purification and properties of L-3,4-dihydroxy-2-butanone-4-phosphate synthase. , 1990, The Journal of biological chemistry.

[8]  A. Bacher,et al.  Biosynthesis of riboflavin. Studies on the reaction mechanism of 6,7-dimethyl-8-ribityllumazine synthase. , 1995, Biochemistry.

[9]  W. Eisenreich,et al.  Biosynthesis of Riboflavin. Stereochemistry of the 3,4-Dihydroxy-2-butanone 4-Phosphate Synthase Reaction , 1998 .

[10]  F. Mȕller.  Chemistry and Biochemistry of Flavoenzymes: Volume I , 1991 .

[11]  A. Bacher,et al.  Design, Synthesis, and Biological Evaluation of Homologous Phosphonic Acids and Sulfonic Acids as Inhibitors of Lumazine Synthase , 1999 .

[12]  R. Huber,et al.  Studies on the lumazine synthase/riboflavin synthase complex of Bacillus subtilis: crystal structure analysis of reconstituted, icosahedral beta-subunit capsids with bound substrate analogue inhibitor at 2.4 A resolution. , 1995, Journal of molecular biology.

[13]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.