The Old Yellow Enzyme family members (OYEs) are NAD(P)H dehydrogenases containing noncovalently bound FMN. These enzymes have been reported in bacteria, yeasts, fungi, plants and in animals. Since 1932, when the first OYE protein was isolated and characterized as the first flavoprotein in general, the physiological role of OYEs is still under discussion. So far, more than 100 structures of OYEs have been published. It is hypothesized that these enzymes may play certain role in cell response to oxidative stress. Moreover, these enzymes have biotechnological and biodegradatory potential and are, therefore, subjects of biochemical and crystalografic studies.
The crystal structure of glycerol trinitrate reductase from Agrobacterium sp. R89-1 (XdpB) has been determined at 2.1 Å resolution in its Apo form. In analogy to proteins homologous to XdpB, the enzyme has the TIM barrel fold with the N-terminal β-hairpin lid. Unexpectedly, XdpB C-terminus interacts with the active and FMN binding sites of a symmetry-related protein. This interaction contributes to stabilization of the crystal lattice and represents a unique structural feature among analogous bacterial enzymes which has possible functional consequences. There was no crystallographic evidence for the presence of natural, noncovalently bound co-factor FMN. The crystallographically well defined C-pentapeptide binds to the amino acid residues lining the FMN site by an extensive network of interactions: 10 hydrogen bonds, 4 water bridges, one CH-pi interaction with the total interaction area is about 86 Å2. Conceptually similar autogenic blocking as reported here has been observed in the crystal structure of an old yellow enzyme OPR3 from the plant species Solanum lycopersicum (PDB code 2hsa (20)).
We expect the same behaviour of other OYE proteins with known 3-D structures because their natural interactions may be disrupted with His tagging of their C-terminuses (PDB 4QNW). Currently we work on detailed explanation of these intermolecular interactions of members of OYE.
The study was supported by grant No. 720414 of the Grant Agency of Charles University, BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF, and by the long-term research development project RVO 61388971 to Institute of Microbiology CAS, v. v. i. and 86 652 036 to Institute of Biotechnology. We thank Dr. M. Šulc for mass spectrometry measurements (Faculty of Science, Charles University, Prague, Czech Republic).
We thank Helmholz Zentrum Berlin for the allocation of synchrotron radiation beamtime.