ribulose-5-phosphate and 3-4-dihydroxy-2-butanone-4-phosphate

Topics: Candida; Carbohydrate Epimerases; Cloning, Molecular; Escherichia coli; Genes, Bacterial; Genes, Fungal; Intramolecular Transferases; Molecular Sequence Data; Recombinant Proteins; Riboflavin; Ribulosephosphates; Substrate Specificity; Sugar Phosphates

3,4-Dihydroxy-2-butanone 4-phosphate is biosynthesized from ribulose 5-phosphate and serves as the biosynthetic precursor for the xylene ring of riboflavin. The gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli has been cloned and sequenced. The gene codes for a protein of 217 amino acid residues with a calculated molecular mass of 23,349.6 Da. The enzyme was purified to near homogeneity from a recombinant E. coli strain and had a specific activity of 1,700 nmol mg-1 h-1. The N-terminal amino acid sequence and the amino acid composition of the protein were in agreement with the deduced sequence. The molecular mass as determined by ion spray mass spectrometry was 23,351 +/- 2 Da, which is in agreement with the predicted mass. The previously reported loci htrP, "luxH-like," and ribB at 66 min of the E. coli chromosome are all identical to the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase, but their role had not been hitherto determined. Sequence homology indicates that gene luxH of Vibrio harveyi and the central open reading frame of the Bacillus subtilis riboflavin operon code for 3,4-dihydroxy-2-butanone 4-phosphate synthase.

Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Carbohydrate Epimerases; Cloning, Molecular; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Heat-Shock Proteins; Intramolecular Transferases; Molecular Sequence Data; Plasmids; Riboflavin; Ribulosephosphates; Sequence Alignment; Sugar Phosphates

The riboflavin precursor, L-3,4-dihydroxy-2-butanone 4-phosphate, is formed from D-ribulose 5-phosphate by a single 24-kDa enzyme. Studies with various specifically 13C-labeled D-ribulose 5-phosphates as substrate showed that the carbon atoms 1-3 of the enzyme product correspond to carbon atoms 1-3 of the substrate, whereas C-4 of the product stems from C-5 of the substrate. Carbon atom 4 of the substrate is released as formate together with the hydrogen atom attached to it. The skeletal rearrangement which leads to the loss of C-4 and the direct linkage between C-3 and C-5 of the substrate is an intramolecular reaction. The hydrogen atom at C-3 of the enzyme product is introduced from solvent water. A reaction mechanism which is in agreement with all experimental data is proposed.

Topics: Candida; Carbohydrate Epimerases; Enzymes; Intramolecular Transferases; Magnetic Resonance Spectroscopy; Mass Spectrometry; Riboflavin; Ribulosephosphates; Sugar Phosphates

The formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione requires a phosphorylated 4-carbon intermediate which has been designated as Compound X (Neuberger, G., and Bacher, A. (1985) Biochem. Biophys. Res. Commun. 127, 175-181). The enzyme catalyzing the formation of Compound X has been purified about 600-fold from the cell extract of the flavinogenic yeast Candida guilliermondii by chromatographic procedures. The purified protein appeared homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of a single polypeptide of 24 kDa. The committed substrate of the enzyme was identified as D-ribulose 5-phosphate. The enzyme yields two products which were identified as L-3,4-dihydroxy-2-butanone 4-phosphate and formate by NMR and CD spectroscopy. Mg2+ is required for activity.

Topics: Candida; Chromatography; Circular Dichroism; Enzymes; Formates; Intramolecular Transferases; Magnesium; Magnetic Resonance Spectroscopy; Molecular Structure; Molecular Weight; Protein Precursors; Pteridines; Riboflavin; Ribulosephosphates; Substrate Specificity; Sugar Phosphates