Crystal structure of FAD synthetase from Corynnebacterium

Crystal structure of FAD synthetase from Corynnebacterium

Crystal
structure of
FAD synthetase
from Corynnebacterium
ammoniagenes: Mecanistic implications.
Beatriz Herguedas1, Marta Martínez-Júlvez1, Susana Frago1, Milagros Medina1, Juan A.
Hermoso2.
1
Departamento de Bioquímica, Biología Molecular y Celular. Instituto de
Biocomputación y Física de Sistemas Complejos. Universidad de Zaragoza
2
Grupo de Cristalografía Macromolecular y Biología Estructural. Instituto de Química
Física Rocasolano, CSIC.
FMN and FAD are essential cofactors of flavoproteins, a large group of proteins
involved in processes like photosynthesis, fatty acid metabolism, mitochondrial electron
transfer chain, apoptosis and DNA repair.
FMN and FAD are synthesized in vivo from the vitamin Riboflavin (RF) in two
sequential steps. The first reaction, catalyzed by Riboflavin kinase (RFK), produces the
phosphorylation of Riboflavin to FMN. In a second process FMN is adenylylated by
FMN-adenylyl transferase (FMNAT) to produce FAD. These reactions are catalyzed by
two different enzymes in eukaryotes, whereas prokaryotes present a bifunctional
enzyme, FAD synthetase, with both activities. The mechanistic and structural
knowledge of these enzymes is very limited.
The bifunctional enzyme of Corynnebacterium ammoniagenes (CaFADS) is being
characterized using site-directed mutagenesis, calorimetry, enzyme activity
measurements, UV-vis spectroscopy and bioinformatic tools [2, 3].
In this study we present the crystal structure of CaFADS. Multi-wavelength Anomalous
Dispersion (MAD) of Selenium atoms in the selenomethionine-labelled protein was
used to solve the phase problem. The selenomethionine-labelled protein was expressed,
purified and crystallized, and the X-Ray Diffraction experiments were carried out at the
BM-16 line of the European Synchrotron Radiation Facility (ESRF).
CaFADS is the first example of a protein from the biosynthetic pathway of FMN and
FAD that has been structurally and functionally characterized. The structure provides
new evidences of the substrate and product binding sites, which will be essential to
propose the structural basis for the catalytic mechanism of the bifunctional FAD
synthetase family.
[1] Efimov I, Kuusk V, Zhang X, McIntire WS. Biochemistry 1998, 37(27):9716-9723.
[2] Susana Frago, Marta Martínez-Júlvez, Ana Serrano and Milagros Medina. BMC
Microbiology, 2008, 8:160.
[3] Susana Frago, Adrián Velázquez-Campoy and Milagros Medina. J. Biol. Chem.,
2009, In press.