Structural analysis of FAD synthetase from Corynebacterium ammoniagenes.
BACKGROUND
The prokaryotic FAD synthetase family - a group of bifunctional enzymes that catalyse riboflavin phosphorylation and FMN adenylylation within a single polypeptide chain- was analysed in terms of sequence and structure.


RESULTS
Sequences of nearly 800 prokaryotic species were aligned. Those related with bifunctional FAD synthetase activities showed conservation of several consensus regions and highly conserved residues. A 3D model for the FAD synthetase from Corynebacterium ammoniagenes (CaFADS) was generated. This model confirms that the N-terminal and C-terminal domains are related to nucleotydyltransferases and riboflavin kinases, respectively. Models for the interaction of CaFADS with its substrates were also produced, allowing location of all the protein substrates in their putative binding pockets. These include two independent flavin binding sites for each CaFADS activity.


CONCLUSIONS
For the first time, the putative presence of a flavin binding site for the adenylylation activity, independent from that related with the phosphorylation activity, is shown. Additionally, these models suggest the functional relevance of some residues putatively involved in the catalytic processes. Their relevant roles were analysed by site-directed mutagenesis. A role was confirmed for H28, H31, S164 and T165 in the stabilisation of the P groups and the adenine moiety of ATP and, the P of FMN for the adenylylation. Similarly, T208, N210 and E268 appear critical for accommodation of the P groups of ATP and the ribityl end of RF in the active site for the phosphorylation process. Finally, the C-terminal domain was shown to catalyse the phosphorylation process on its own, but no reaction at all was observed with the individually expressed N-terminal domain.
Frago
Susana
S
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias and Institute of Biocomputation and Physics of Complex Systems, Universidad de Zaragoza, Zaragoza, Spain. sfrago@unizar.es
Martínez-Júlvez
Marta
M
Serrano
Ana
A
Medina
Milagros
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005486:Flavin Mononucleotide / Q000096:biosynthesis; D005182:Flavin-Adenine Dinucleotide / Q000096:biosynthesis; D005415:Flavins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D009713:Nucleotidyltransferases; C020143:FMN adenylyltransferase