The Active Site Base Controls Cofactor Reactivity inEscherichia coliAmine Oxidase:  X-ray Crystallographic Studies with Mutational Variants^,

Abstract

Amine oxidases utilize a proton abstraction mechanism following binding of the amine substrate to the C5 position of the cofactor, the quinone form of trihydroxyphenylalanine (TPQ). Previous work [Wilmot, C. M., et al. (1997) Biochemistry 36, 1608-1620] has shown that Asp383 in Escherichia coliamine oxidase (ECAO) is the catalytic base which performs the key step of proton abstraction. This paper explores in more depth this and other roles of Asp383. The crystal structures of three mutational variants are presented together with their catalytic properties, visible spectra, and binding properties for a substrate-like inhibitor, 2-hydrazinopyridine (2-HP), in comparison to those of the wild type enzyme. In wild type ECAO, the TPQ is located in a wedge-shaped pocket which allows more freedom of movement at the substrate binding position (C5) than for TPQ ring carbons C1-C4. A role of Asp383, whose carboxylate is located close to O5, is to stabilize the TPQ in its major conformation in the pocket. Replacement of Asp383 with the isostructural, but chemically distinct, Asn383 does not affect the location or dynamics of the TPQ cofactor significantly, but eliminates catalytic activity and drastically reduces the affinity for 2-HP. Removal of the side chain carboxyl moiety, as in Ala383, additionally allows the TPQ the greater conformational flexibility to coordinate to the copper, which demonstrates that Asp383 helps maintain the active site structure by preventing TPQ from migrating to the copper. Glu383 has a greatly decreased catalytic activity, as well as a decreased affinity for 2-HP relative to that of wild type ECAO. The electron density reveals that the longer side chain of Glu prevents the pivotal motion of the TPQ by hindering its movement within the wedge-shaped active site pocket. The results show that Asp383 performs multiple roles in the catalytic mechanism of ECAO, not only in acting as the active site base at different stages of the catalytic cycle but also in regulating the mobility of the TPQ that is essential to catalysis.

Biodegradation of Aromatic Compounds by Escherichia coli

“…The structure of MaoA has been determined crystallographically, and each subunit consists of four distinct domains with the active site being located in the C-terminal one (206,230). The central water-filled cavity of the dimer interface is proposed to be the entry site for molecular oxygen.…”

“…The copper is coordinated by three conserved histidine residues and two water molecules. The TPQ is close to the copper and appears to have high rotational mobility (206,265). The catalytic reaction of the enzyme divides into a reductive half-reaction and an oxidative half-reaction.…”

“…The oxidative half-reaction involves the copper ion and molecular oxygen and recycles the reduced enzyme back to its oxidized resting state, accompanied by the release of ammonia and hydrogen peroxide. The Asp-383 residue performs multiple roles in the catalytic mechanism, acting not only as the active-site base at different stages of the catalytic cycle but also in regulating the mobility and regenerating the TPQ cofactor that is essential to catalysis (206,336).…”

Probing the Catalytic Mechanism of Copper Amine Oxidase from Arthrobacter globiformis with Halide Ions

“…This suggests that the equilibrium between TPQ amr ⅐Cu(II) and TPQ sq ⅐Cu(I) is accompanied by a substantial conformational reorganization of the TPQ ring. Indeed, the conformational flexibility of TPQ within the active site of CAOs has previously been suggested to be important in catalysis (24,30,31). In the subsequent oxidative half-reaction, the reduced cofactor is reoxidized by dioxygen to produce hydrogen peroxide and an iminoquinone intermediate (TPQ imq ), which is further hydrolyzed to form the oxidized cofactor, releasing ammonia in the following step (1)(2)(3).…”

Synthesis and evaluation of resins bearing substrate-like inhibitor functions for capturing copper amine oxidases

“…Knowledge concerning the physiological role, mechanism of action [1][2][3][4] and numerous X-ray structures [5][6][7][8][9][10][11][12][13][14] of these enzymes, as well as methods for protein expression of CAOs, 15 making larger quantities of protein samples available, which is useful for various experiments, has grown progressively in the past two decades.…”