Open AccessSynthesis, Characterization and Preliminary Crystallographic Data of N 6 ‐(6‐carbamoylhexyl)‐FAD‐ d ‐amino‐acid Oxidase from Pig Kidney, a Semi‐Synthetic Oxidase
Author(s) -
Stocker Achim,
Hecht HansJürgen,
Bückmann Andreas F.
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.0519z.x
Subject(s) - d amino acid oxidase , oxidase test , chemistry , cofactor , moiety , amino acid , stereochemistry , covalent bond , enzyme , biochemistry , organic chemistry
The FAD analogue, N 6 ‐(6‐carboxyhexyl)‐FAD, carrying a hexanoic acid residue at the N 6 position of the adenine moiety was synthesized. A new semi‐synthetic oxidase, N 6 ‐(6‐carbamoylhexyl)‐FAD‐ d ‐amino acid oxidase, was prepared by reacting the succinimido ester of N 6 ‐(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 N 6 ‐(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 N 6 ‐(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, V max (app) = 17.7 μmol min ‐1 mg ‐1 ; K M (app) = 4.5 mM; native holo‐DAAO, V max = 12.2 μmol min ‐1 mg ‐1 ; K M = 1.8 mM). Compared with the native holo‐ d ‐amino acid oxidase, this new semi‐synthetic N 6 ‐(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 N 6 ‐(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.