Affiliation:
1. Institut für Technische Mikrobiologie, Hochschule Mannheim, Windeckstr. 110, 68163 Mannheim, Germany
2. Eurofins Medigenomix GmbH, Fraunhoferstr. 22, 82152 Martinsried, Germany
Abstract
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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
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