Consistent with this suggestion, we have found that oligonucleotide derivatives containing lipophilic substituents bound with substantially increased affinity to their single-stranded complementary oligonucleotides (Jagcr, A.; Levy, M. J.; Hecht, S. M., in preparation).
The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits surrounding a core of 3 alpha subunits. The beta subunits catalyze the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with (3S)-3,4-dihydroxy-2-butanone under formation of 6,7-dimethyl-8-ribityllumazine. This intermediate is converted to riboflavin by the alpha subunits via an unusual dismutation yielding 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione as second product. (3R)- and (3S)-3,4-dihydroxy-2-butanone 4-phosphate were synthesized. Both enantiomers can serve as substrate for 6,7-dimethyl-8-ribityllumazine synthase. The reaction rate of the natural S-enantiomer is about 6-fold higher than that of the R-enantiomer. The Km value for (3S)-3,4-dihydroxy-2-butanone 4-phosphate is 130 microM, and the Km value for the pyrimidine substrate is 5 microM. Diacetyl and 3,4-dihydroxy-2-butanone 3-phosphate do not serve as substrates for lumazine synthase. The enzyme-catalyzed condensation of the carbohydrate with the pyrimidine is strictly regiospecific. The enzyme does not catalyze the exchange of protons between (3S)-3,4-dihydroxy-2-butanone 4-phosphate and solvent water in the absence of the pyrimidine cosubstrate. A reaction mechanism starting with the formation of a Schiff base followed by elimination of phosphate and cyclization is proposed. The lumazine synthase activities of the native enzyme complex and of reconstituted, hollow beta 60 capsids are virtually identical (about 12,000 nmol mg-1 h-1).
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-' h-'. 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, "IuxH-like," and nibB 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 BaciUlus subtilis riboflavin operon code for 3,4-dihydroxy-2-butanone 4-phosphate synthase.
GTP cyclohydrolase II catalyzes the first committed step in the biosynthesis of riboflavin. The gene coding for this enzyme in Escherichia coli has been cloned by marker rescue. Sequencing indicated an open reading frame of 588 bp coding for a 21.8-kDa peptide of 196 amino acids. The gene was mapped to a position at 28.2 min on the E. coli chromosome and is identical with ribA. GTP cyclohydrolase II was overexpressed in a recombinant strain carrying a plasmid with the cloned gene. The enzyme was purified to homogeneity from the recombinant strain. The N-terminal sequence determined by Edman degradation was identical to the predicted sequence. The sequence is homologous to the 3' part of the central open reading frame in the riboflavin operon of Bacillus subtilis.
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