Revisitation of the βCl-Elimination Reaction of d-Amino Acid Oxidase

Abstract

, which was later confirmed by others (2-4). Based on this, the mechanism depicted in Scheme 1 for the elimination reaction was put forward (1). In this reaction the substrate ␣H as H ϩ is abstracted to form a carbanion intermediate, which then releases Cl Ϫ to yield the final products pyruvate and NH 4 ϩ . In this reaction the redox state of the flavin cofactor remains unaltered."Normal" dehydrogenation of ␤Cl-D-Ala would yield ␤Cl-pyruvate (␤Cl-Py) as the final product and lead to reduction of the flavin cofactor (5). The initial experiment (1) provided the first clues for formulating a general concept for the mechanism underlying flavin dehydrogenation and represents the birth of the so-called carbanion mechanism. This concept then gained wide acceptance for decades to come. However, some doubts soon emerged (2, 6 -8); experiments by Hersh and Jorns (9) demonstrated that for a DAAO in which the flavin cofactor was replaced by 5-deaza-flavin, Cl Ϫ was not eliminated. This puzzle remained unanswered for a long time, as the flavin ought not to be involved directly in elimination. Later experiments based on the concept of the linear free energy relationship were not in favor of the carbanion mechanism either (10, 11). A way out of the conundrum emerged when the three-dimensional structure of two related DAAOs was identified (12, 13); these studies showed unambiguously that there is no functional group at the active center of the enzyme that could act as a base in abstracting the substrate ␣H as H ϩ . Furthermore, the structures of complexes of Rhodotorula gracilis D-amino acid oxidase (RgDAAO) with D-alanine or D-CF 3 -alanine show that the substrate ␣C-H points directly toward

Further evidence in favour of a carbanion mechanism for glycolate oxidase

“…This reaction does not in itself constitute an absolute proof. Indeed, it was suggested that halogen elimination could occur after enzyme reduction, when a hydride would displace the halogen from the normal keto acid product, as has been shown for d ‐amino acid oxidase [ 87 ]. However, a number of studies on Fcb2 WT and variant forms provided significant mechanistic information.…”

Multiple functionalities of reduced flavin in the non-redox reaction catalyzed by UDP-galactopyranose mutase

“…N5-adducts have also been proposed in the reaction of monoamine oxidase with several substrates and inhibitors [22]. The β-Cl-elimination activity of D-amino acid oxidase has been proposed to occur via formation of a transient N5-covalent intermediate [23]. In these examples, after the initial attack of the substrate/analogs, formation of a N5-iminium ion or decay of the flavin N5-adduct is coupled to an elimination step (e.g., nitrite, chloride) (Scheme 2).…”

D-Amino Acids and Their Oxidase in Farm Animals — The Role and Opportunities

“…In mammals, it is found in brain, kidneys, liver, lungs, etc. It is remarkable that DAAO concentration in liver and kidneys of pigs is particularly high [7]. Multiple enzyme functions indicate the relation between changes in DAAO activity in the cell with different regulatory processes in the body [8,9].…”