Synthesis, Characterization and Preliminary Crystallographic Data of N6‐(6‐carbamoylhexyl)‐FAD‐d‐amino‐acid Oxidase from Pig Kidney, a Semi‐Synthetic Oxidase
Open Access
- 1 June 1996
- journal article
- Published by Wiley in European Journal of Biochemistry
- Vol. 238 (2) , 519-528
- https://doi.org/10.1111/j.1432-1033.1996.0519z.x
Abstract
The FAD analogue, N6‐(6‐carboxyhexyl)‐FAD, carrying a hexanoic acid residue at the N6 position of the adenine moiety was synthesized. A new semi‐synthetic oxidase, N6‐(6‐carbamoylhexyl)‐FAD‐d‐amino acid oxidase, was prepared by reacting the succinimido ester of N6‐(6‐carboxyhexyl)‐FAD with apo‐d‐amino‐acid oxidase from pig kidney in the presence of benzoate. Reaction conditions and methods have been developed for preparing pure semi‐synthetic and fully active N6‐(6‐carbamoylhexyl)‐FAD‐d‐amino acid oxidase that contains 1 covalently bound FAD analogue/subunit, as verified by redialysis, ultraviolet spectrophotometry, electrospray ionization (ESI)‐MS and peptide mapping.Presumably, the N6‐(6‐carbamoylhexyl)‐FAD moiety of this semi‐synthetic d‐amino‐acid oxidase (DAAO), selectively bound to Lys163, has a structurally similar position to that of the non‐covalently bound FAD of the native holoenzyme, since both DAAO forms show very similar kinetic properties (semi‐synthetic DAAO, Vmax(app) = 17.7 μmol min‐1 mg‐1; KM(app) = 4.5 mM; native holo‐DAAO, Vmax= 12.2 μmol min‐1 mg‐1; KM= 1.8 mM). Compared with the native holo‐d‐amino acid oxidase, this new semi‐synthetic N6‐(6‐carbamoylhexyl)‐FAD‐d‐amino acid oxidase is a considerably more stable enzyme that shows meso‐thermostability and withstands inactivation on dilution. Probably, the lack of dissociation of FAD and, consequently, the absence of the instable apoenzyme are responsible for these phenomena. Preliminary investigations resulted in finding convenient and reproducible crystallization conditions for N6‐(6‐carbamoylhexyl)‐FAD‐d‐amino acid oxidase. The single crystals, obtained by the sitting‐drop method using ammonium sulfate as precipitant, belong to the tetragonal space group I422 with cell dimensions a= 16.3 nm, c= 13.6 nm. The crystals diffract to 0.3‐nm resolution, with two molecules being present in the asymmetric unit, demonstrating the two‐subunit quarternary structure of this semi‐synthetic D‐amino‐acid oxidase.Keywords
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