BIOSYNTHESIS OF RIBOFLAVIN - AN UNUSUAL RIBOFLAVIN SYNTHASE OF METHANOBACTERIUM-THERMOAUTOTROPHICUM

Citation
S. Eberhardt et al., BIOSYNTHESIS OF RIBOFLAVIN - AN UNUSUAL RIBOFLAVIN SYNTHASE OF METHANOBACTERIUM-THERMOAUTOTROPHICUM, Journal of bacteriology, 179(9), 1997, pp. 2938-2943
Citations number
34
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
ISSN journal
0021-9193
Volume
179
Issue
9
Year of publication
1997
Pages
2938 - 2943
Database
ISI
SICI code
0021-9193(1997)179:9<2938:BOR-AU>2.0.ZU;2-7
Abstract
Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermo-autotrophicum. The enzyme had a spe cific activity of about 2,700 nmol mg(-1) h(-1) at. 65 degrees C, whic h is relatively low compared to those of riboflavin synthases of eubac teria and yeast, Amino acid sequences obtained after proteolytic cleav age had no similarity with known riboflavin synthases, The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli. The ribC gene of M. thermoautotrophicum specifies a protein of 153 amino acid residues . The predicted amino acid sequence agrees with the information gleane d from Edman degradation of the isolated protein and shows 67% identit y with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii. The ribC gene is adjacent to a cluster o f four genes with similarity to the genes cbiMNOO of Salmonella typhim urium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B-12). The amino ac id sequence predicted by the ribC gene of M. thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast. Most notably, the M. thermoautotrophicum protein does not show the internal sequence homology characteristic of eubact erial and yeast riboflavin synthases. The protein of M. thermoautotrop hicum can be expressed efficiently in a recombinant E. coli strain, Th e specific activity of the purified, recombinant protein is 1,900 nmol mg(-1) h(-1) at 65 degrees C. In contrast to riboflavin synthases fro m eubacteria and fungi, the methanobacterial enzyme has an absolute re quirement for magnesium ions, The 5' phosphate of 6,7-dimethyl-8-ribit yllumazine does not act as a substrate, The findings suggest that ribo flavin synthase has evolved independently in eubacteria and methanobac teria.