Exploring the Roles of the Metal Ions in Escherichia coli Copper Amine Oxidase,

To investigate the role of the active site copper in Escherichia coli copper amine oxidase (ECAO), we initiated a metal-substitution study. Copper reconstitution of ECAO (Cu-ECAO) restored only ∼12% wild-type activity as measured by kcat(amine). Treatment with EDTA, to remove exogenous divalent metals, increased Cu-ECAO activity but reduced the activity of wild-type ECAO. Subsequent addition of calcium restored wild-type ECAO and further enhanced Cu-ECAO activities. Cobalt-reconstituted ECAO (Co-ECAO) showed lower but significant activity. These initial results are consistent with a direct electron transfer from TPQ to oxygen stabilized by the metal. If a Cu(I)-TPQ semiquinone mechanism operates, then an alternative outer-sphere electron transfer must also exist to account for the catalytic activity of Co-ECAO. The positive effect of calcium on ECAO activity led us to investigate the peripheral calcium binding sites of ECAO. Crystallographic analysis of wild-type ECAO structures, determined in the presence and absence of EDTA, confirmed that calcium is the normal ligand of these peripheral sites. The more solvent exposed calcium can be easily displaced by mono- and divalent cations with no effect on activity, whereas removal of the more buried calcium ion with EDTA resulted in a 60−90% reduction in ECAO activity and the presence of a lag phase, which could be overcome under oxygen saturation or by reoccupying the buried site with various divalent cations. Our studies indicate that binding of metal ions in the peripheral sites, while not essential, is important for maximal enzymatic activity in the mature enzyme

Amine oxidase substrates for impaired glucose tolerance correction

Amine oxidases are widely distributed from microorganisms to vertebrates and produce hydrogen peroxide plus aldehyde when catabolizing endogenous or xenobiotic amines. Novel roles have been attributed to several members of the amine oxidase families, which cannot be more considered as simple amine scavengers. Semicarbazide-sensitive amine oxidase (SSAO) is abundantly expressed in mammalian endothelial, smooth muscle, and fat cells, and plays a role in lymphocyte adhesion to vascular wall, arterial fiber elastic maturation, and glucose transport, respectively. This latter role is detailed and the perspectives opened by the insulin-like actions of amine oxidase substrates are discussed in the present review. Independent studies have demonstrated that SSAO substrates and monoamine oxidase substrates mimic diverse insulin effects in adipocytes: glucose transport activation, lipogenesis stimulation and lipolysis inhibition. These substrates also stimulate in vitro adipogenesis. Acute in vivo administration of amine oxidase substrates improves glucose tolerance in rats, mice and rabbits, while chronic treatments with benzylamine plus vanadate exert an antihyperglycaemic effect in diabetic rats. Dietary supplementations with methylamine, benzylamine or tyramine have been evidenced to influence metabolic control in rodents by increasing glucose tolerance or decreasing lipid mobilisation, without noticeable changes in the plasma markers of lipid peroxidation or protein glycation, despite adverse effects on vasculature. Thus, the ingested amines are not totally degraded at the intestinal barreer and can act on adipose and vascular tissues. In regard with this influence on metabolic control, more attention must be paid to the composition or supplementation in amines in foods and nutraceutics