The Bifunctional Flavokinase/Flavin Adenine Dinucleotide Synthetase from Streptomyces davawensis Produces Inactive Flavin Cofactors and Is Not Involved in Resistance to the Antibiotic Roseoflavin▿

摘要

Streptomyces davawensis 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. davawensis, which would not allow the formation of toxic RoFMN/RoFAD. The gene ribC from S. davawensis 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, kcat/Km = 1.7 10−2 μM−1 s−1; riboflavin, kcat/Km = 7.5 10−3 μM−1 s−1). Similar results were obtained for RibC from the roseoflavin-sensitive bacterium Bacillus subtilis (roseoflavin, kcat/Km = 1.3 10−2 μM−1 s−1; riboflavin, kcat/Km = 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.