Synthesis, Characterization and Preliminary Crystallographic Data of N⁶‐(6‐carbamoylhexyl)‐FAD‐d‐amino‐acid Oxidase from Pig Kidney, a Semi‐Synthetic Oxidase

Abstract

The FAD analogue, Nh-(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-~arbamoylhexyl)-FAD-~-arnino acid oxidase, was prepared by reacting the succinimido ester of N6-(6-carboxyhexyl)-FAD with apo-Damino-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-~-amino acid oxidase that contains 1 covalently bound FAD analoguehbunit, as verified by redialysis, ultraviolet spectrophotometry, electrospray ionization (ES1)-MS and peptide mapping.Presumably, the Nb-(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,,,(app) = 17.7 pmol min-' mg-'; K,(app) = 4.5 mM; native holo-DAAO, V,,,,, = 12.2 pmol min-' mg-'; KM = 1.8 mM). Compared with the native holo-D-amino acid oxidase, this new semi-synthetic N6-(6-carbamoylhexyl)-FAD-~-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 Nh-(6-carbamoylhexyI)-FAD-~-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 u = 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.

Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: the shape of the active-site cavity controls substrate specificity

“…The covalent linkage of the FAD cofactor to the protein is a feature common to most members of the VAO-related oxidoreductase family [7]. Although covalent flavinylation has been observed in many flavoproteins, the mechanism of formation and the functional role of the covalent bond are poorly understood [10,11]. In this respect, the VAOrelated oxidoreductases are particularly attractive for study as they portray different types of flavinylation, such as the 8␣-(O-tyrosyl)-FAD of PCMH, the 8α-(N1-histidyl)-FAD of L-gulono-γ-lactone oxidase and the 8α-(N3-histidyl)-FAD of VAO and 6-hydroxy-D-nicotine oxidase.…”

Crystallization and preliminary x-ray analysis of the flavoenzyme vanillyl-alcohol oxidase fromPenicillium Simplicissimum

“…Such an approach has been successfully used in the case of D-amino acid oxidase and it is likely to be applicable to other flavoenzymes. 7 In the framework of a project devoted to the structural characterization of FAD-dependent oxidases, 8 we have undertaken the X-ray analysis of VAO. Knowledge of the structure will provide the basis for the understanding of the novel mechanistic properties of this enzyme and will shed light into the structural and functional role of the covalent attachment of the FAD cofactor.…”

Engineering the Properties of D-Amino Acid Oxidases by a Rational and a Directed Evolution Approach

“…In 1996, Buckmann and coworkers [95] prepared a new semisynthetic oxidase by reacting the succimidoester of N 6 -(6-carboxyhexyl)-FAD, a very peculiar site-directed reagent for probing the flavin-binding site of flavoproteins, with the pkDAAO apoprotein. The fully active N 6 -(6-carbamoylhexyl)-FAD DAAO contains one covalently bound FAD analogue per subunit attached to Lys163, yielding a semisynthetic pkDAAO whose intrinsic stability is significantly increased and that could possibly help develop more robust devices [95]. The covalent conjugation of the FAD analogue converts pkDAAO into a meso-thermostable enzyme, thus suggesting that the main factor for thermostabilization could be the full coenzyme stabilization related to the absence of the apoprotein form (see the Stability Section).…”