The Bifunctional Flavokinase/Flavin Adenine Dinucleotide Synthetase from Streptomyces davawensis Produces Inactive Flavin Cofactors and Is Not Involved in Resistance to the Antibiotic Roseoflavin
- 1 March 2008
- journal article
- research article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 190 (5), 1546-1553
- https://doi.org/10.1128/jb.01586-07
Abstract
Streptomyces davawensi s synthesizes the antibiotic roseoflavin, one of the few known natural riboflavin analogs, and is roseoflavin resistant. It is thought that the endogenous flavokinase (EC 2.7.1.26)/flavin adenine dinucleotide (FAD) synthetase (EC 2.7.7.2) activities of roseoflavin-sensitive organisms are responsible for the antibiotic effect of roseoflavin, producing the inactive cofactors roseoflavin-5′-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) from roseoflavin. To confirm this, the FAD-dependent Sus scrofa d -amino acid oxidase (EC 1.4.3.3) was tested with RoFAD as a cofactor and found to be inactive. It was hypothesized that a flavokinase/FAD synthetase (RibC) highly specific for riboflavin may be present in S. davawensi s, which would not allow the formation of toxic RoFMN/RoFAD. The gene ribC from S. davawensi s was cloned. RibC from S. davawensis was overproduced in Escherichia coli and purified. Analysis of the flavokinase activity of RibC revealed that the S. davawensis enzyme is not riboflavin specific (roseoflavin, k cat / K m = 1.7 10 −2 μM −1 s −1 ; riboflavin, k cat / K m = 7.5 10 −3 μM −1 s −1 ). Similar results were obtained for RibC from the roseoflavin-sensitive bacterium Bacillus subtilis (roseoflavin, k cat / K m = 1.3 10 −2 μM −1 s −1 ; riboflavin, k cat / K m = 1.3 10 −2 μM −1 s −1 ). Both RibC enzymes synthesized RoFAD and RoFMN. The functional expression of S. davawensis ribC did not confer roseoflavin resistance to a ribC -defective B. subtilis strain.Keywords
This publication has 42 references indexed in Scilit:
- BRENDA, AMENDA and FRENDA: the enzyme information system in 2007Nucleic Acids Research, 2007
- The riboflavin kinase encoding generibRofBacillus subtilisis a part of a 10 kb operon, which is negatively regulated by theyrzCgene productFEMS Microbiology Letters, 2005
- Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilisNature Biotechnology, 2003
- Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2)Nature, 2002
- Macrolide Resistance Gene mreA of Streptococcus agalactiae Encodes a FlavokinaseAntimicrobial Agents and Chemotherapy, 2001
- Gapped BLAST and PSI-BLAST: a new generation of protein database search programsNucleic Acids Research, 1997
- Formation of Roseoflavin from 8-Amino- and 8-Methylamino-8-Demethyl-D-RiboflavinThe Journal of Biochemistry, 1987
- Preparation and properties of immobilized flavokinaseBiotechnology & Bioengineering, 1979
- Chemical and enzymic properties of riboflavin analogsBiochemistry, 1978
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976