The role of cofactor binding in tryptophan accessibility and conformational stability of His-tagged D-amino acid oxidase from Trigonopsis variabilis.

d-amino acid oxidase from Trigonopsis variabilis (TvDAAO) is a flavoenzyme with high biotechnological and industrial interest. The overexpression and purification of the apoprotein form of a recombinant His-tagged TvDAAO allowed us to go deep into the structural differences between apoenzyme and holoenzyme, and on the cofactor binding and its contribution to enzyme stability. A significant decrease in intrinsic fluorescence emission took place upon FAD binding, associated to cofactor induced conformational transitions or subunit dimerization that could affect the local environment of protein tryptophan residues. Furthermore, acrylamide-quenching experiments indicated that one of the five tryptophan residues of TvDAAO became less accessible upon FAD binding. A K(d)=1.5+/-0.1x10(-7) M for the dissociation of FAD from TvDAAO was calculated from binding experiments based on both quenching of FAD fluorescence and activity titration curves. Secondary structure prediction indicated that TvDAAO is a mixed alpha/beta protein with 8 alpha-helices and 14 beta-sheets connected by loops. Prediction results were in good agreement with the estimates obtained by circular dichroism which indicated that both the apoenzyme and the holoenzyme had the same structural component ratios: 34% alpha-helix content, 20% beta-structure content (14% antiparallel and 6% parallel beta-sheet), 15% beta-turns and 31% of random structure. Circular dichroism thermal-transition curves suggested single-step denaturation processes with apparent midpoint transition temperatures (T(m)) of 37.9 degrees C and 41.4 degrees C for the apoenzyme and the holoenzyme, respectively. A three-dimensional model of TvDAAO built by homology modelling and consistent with the spectroscopic studies is shown. Comparing our results with those reported for pig kidney (pkDAAO) and Rhodotorula gracilis (RgDAAO) d-amino acid oxidases, a "head-to-head" interaction between subunits in the TvDAAO dimer might be expected.