Molecular characterization of FMN1, the structural gene for the monofunctional flavokinase of Saccharomyces cerevisiae

Flavokinase catalyzes the transfer of the gamma-phosphoryl group of ATP to riboflavin to form the flavocoenzyme FMN. Consistent patterns of sequence s imilarities have identified the open reading frame of unknown function YDR2 36c as a candidate to encode flavokinase in Saccharomyces cerevisiae, In or der to determine whether the product of this gene corresponds to yeast flav okinase, its coding region was amplified from S, cerevisiae genomic DNA by polymerase chain reaction and expressed in Escherichia coli, The purified f orm of the expressed recombinant protein efficiently catalyzed the formatio n of FMN from riboflavin and ATP, In contrast to bifunctional prokaryotic f lavokinase/FAD synthetase enzymes, the yeast enzyme did not show accompanyi ng FAD synthetase activity. Deletion of YDR236c produced yeast mutants unab le to grow on rich medium; however, the growth of the ydr236c Delta mutants could be rescued by the addition of FMN to the medium. Overexpression of Y DR236e caused a 50-fold increase in flavokinase specific activity in yeast cells. These findings demonstrate that YDR236e corresponds to the gene enco ding a monofunctional flavokinase in yeast, which we propose to be designat ed as FMN1. The FMN1 gene codes for a 25-kDa protein with characteristics o f signals for import into mitochondria. By immunoblotting analysis of Sacch aromyces subcellular fractions, we provide evidence that the Fmn1 protein i s localized in microsomes and in mitochondria, Analysis of submitochondrial fractions revealed that the mitochondrial form of Fmn1p is an integral pro tein 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 eukaryot ic organisms such as Schizosaccharomyces pombe, Arabidopsis thaliana, Droso phila melanogaster, Caenorhabditis elegans, and humans.