NUCLEOTIDE-SEQUENCE OF THE FAD SYNTHETASE GENE FROM CORYNEBACTERIUM AMMONIAGENES AND ITS EXPRESSION IN ESCHERICHIA-COLI

The nucleotides of a bifunctional enzyme FAD synthetase gene, which showed both flavokinase and ATP:FMN adenylyltransferase activities, from Corynebacterium ammoniagenes were sequenced. The FAD synthetase gene product consisted of 338 amino acids and had a calculated molecular weight of 37,712. The deduced protein sequence of the FAD synthetase shared a homology with those of the protein X of Escherichia coli, which has been reported to have both flavokinase and ATP:FMN adenylyltransferase activities like the FAD synthetase of C. ammoniagenes, and the protein X of Pseudomonas fluorescens. From the analysis of the flanking sequences of the FAD synthetase gene, the gene organization and the operon structure around the FAD synthetase gene of C. ammoniagenes were thought to be different from those of Gram-negative bacteria. An over-expression system of the FAD synthetase of C, ammoniagenes was constructed in E, coli to study the structure and function of the protein, Under the tandem tryptophan promoter, the FAD synthetase activity increased 2231 times compared to that of non-transformed C. ammoniagenes.