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J. Biol. Chem., Vol. 275, Issue 37, 28618-28624, September 15, 2000
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From the Departamento de Microbiología y Genética,
CSIC/Universidad de Salamanca, Campus Miguel de Unamuno,
37007 Salamanca, Spain
Flavokinase catalyzes the transfer of the
Molecular Characterization of FMN1, the Structural
Gene for the Monofunctional Flavokinase of Saccharomyces
cerevisiae*
, and
-phosphoryl group of ATP to riboflavin to form the flavocoenzyme
FMN. Consistent patterns of sequence similarities have identified the
open reading frame of unknown function YDR236c as a
candidate to encode flavokinase in Saccharomyces
cerevisiae. In order to determine whether the product of this
gene corresponds to yeast flavokinase, its coding region was amplified
from S. cerevisiae genomic DNA by polymerase chain reaction
and expressed in Escherichia coli. The purified form of the
expressed recombinant protein efficiently catalyzed the formation of
FMN from riboflavin and ATP. In contrast to bifunctional prokaryotic
flavokinase/FAD synthetase enzymes, the yeast enzyme did not show
accompanying FAD synthetase activity. Deletion of YDR236c
produced yeast mutants unable to grow on rich medium; however, the
growth of the ydr236c
mutants could be rescued by the
addition of FMN to the medium. Overexpression of YDR236c
caused a 50-fold increase in flavokinase specific activity in yeast
cells. These findings demonstrate that YDR236c corresponds
to the gene encoding a monofunctional flavokinase in yeast, which we
propose to be designated as FMN1. The FMN1 gene
codes for a 25-kDa protein with characteristics of signals for import
into mitochondria. By immunoblotting analysis of
Saccharomyces subcellular fractions, we provide evidence
that the Fmn1 protein is localized in microsomes and in mitochondria.
Analysis of submitochondrial fractions revealed that the mitochondrial
form of Fmn1p is an integral protein of the inner membrane exposing its
COOH-terminal domain to the matrix space. A similarity search in the
data base banks revealed the presence of sequences homologous to yeast
flavokinase in the genome of several eukaryotic organisms such as
Schizosaccharomyces pombe, Arabidopsis
thaliana, Drosophila melanogaster,
Caenorhabditis elegans, and humans.
*
This work was supported in part by BASF AG and Grant
1FD97-2131 from the Comisión Interministerial de Ciencia y
Tecnología (CICYT), Spain.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by fellowship from the Junta de Castilla y León, Spain.
§
To whom correspondence should be addressed. Tel.: 34-923-294671;
Fax: 34-923-224876; E-mail: revuelta@gugu.usal.es.
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