Identification of human and rat FAD-AMP lyase (cyclic FMN forming) as ATP-dependent dihydroxyacetone kinases.
Rat liver FAD-AMP lyase or FMN cyclase is the only known enzymatic source of the unusual flavin nucleotide riboflavin 4',5'-cyclic phosphate. To determine its molecular identity, a peptide-mass fingerprint of the purified rat enzyme was obtained. It pointed to highly related, mammalian hypothetical proteins putatively classified as dihydroxyacetone (Dha) kinases due to weaker homologies to biochemically proven Dha kinases of plants, yeasts, and bacteria. The human protein LOC26007 cDNA was used to design PCR primers. The product amplified from human brain cDNA was cloned, sequenced (GenBank Accession No. ), and found to differ from protein LOC26007 cDNA by three SNPs. Its heterologous expression yielded a protein active both as FMN cyclase and ATP-dependent Dha kinase, each activity being inhibited by the substrate(s) of the other. Cyclase and kinase activities copurified from rat liver extracts. Evidence supports that a single protein sustains both activities, probably in a single active center. Putative Dha kinases from other mammals are likely to be FMN cyclases too. Future work will profit from the availability of the structure of Citrobacter freundii Dha kinase, which contains substrate-interacting residues conserved in human Dha kinase/FMN cyclase.
Cabezas
Alicia
A
Unidad de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Extremadura, 06080 Badajoz, Spain.
Costas
María Jesús
MJ
Pinto
Rosa María
RM
Couto
Ana
A
Cameselle
José Carlos
JC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004098:Dihydroxyacetone / Q000494:pharmacology; D005486:Flavin Mononucleotide / Q000096:biosynthesis; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D019761:Phosphorus-Oxygen Lyases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment
D011994:Recombinant Proteins; C111331:riboflavin 4',5'-cyclic phosphate; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C023132:glycerone kinase; D019761:Phosphorus-Oxygen Lyases; C111330:FMN cyclase; D004098:Dihydroxyacetone