SYNTHESIS, CHARACTERIZATION AND PRELIMINARY CRYSTALLOGRAPHIC DATA OF N-6-(6-CARBAMOYLHEXYL)-FAD-D-AMINO-ACID OXIDASE FROM PIG-KIDNEY, A SEMISYNTHETIC OXIDASE

The FAD analogue, N-6-(6-carboxyhexyl)-FAD, carrying a hexanoic acid r esidue at the N-6 position of the adenine moiety was synthesized. A ne w semi-synthetic oxidase, N-6-(6-carbamoylhexyl)-FAD-D-amino acid oxid ase, was prepared by reacting the succinimido ester of N-6-(6-carboxyh exyl)-FAD with apo-D-amino-acid oxidase from pig kidney in the presenc e of benzoate. Reaction conditions and methods have been developed for preparing pure semi-synthetic and fully active N-6-(6-carbamoylhexyl) -FAD-D-amino acid oxidase that contains 1 covalently bound FAD analogu e/subunit, as verified by redialysis, ultraviolet spectrophotometry, e lectrospray ionization (ESI)-MS and peptide mapping. Presumably, the N -6-(6-carbamoylhexyl)-FAD moiety of this semi-synthetic D-amino-acid o xidase (DAAO), selectively bound to Lys163, has a structurally similar position to that of the non-covalently bound FAD of the native holoen zyme, since both DAAO forms show very similar kinetic properties (semi -synthetic DAAO, V-max(app) = 17.7 mu mol min(-1) mg(-1); K-M(app) = 4 .5 mM; native holo-DAAO, V-max = 12.2 mu mol min(-1) mg(-1); K-M = 1.8 mM). Compared with the native holo-D-amino acid oxidase, this new sem i-synthetic N-6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase is a consi derably more stable enzyme that shows meso-thermostability and withsta nds inactivation on dilution. Probably, the lack of dissociation of FA D and, consequently, the absence of the instable apoenzyme are respons ible for these phenomena. Preliminary investigations resulted in findi ng convenient and reproducible crystallization conditions for N-6-(6-c arbamoylhexyl)-FAD-D-amino acid oxidase. The single crystals, obtained by the sitting-drop method using ammonium sulfate as precipitant, bel ong to the tetragonal space group 1422 with cell dimensions a = 16.3 n m, c = 13.6 nm. The crystals diffract to 0.3-nm resolution, with two m olecules being present in the asymmetric unit, demonstrating the two-s ubunit quarternary structure of this semi-synthetic D-amino-acid oxida se.