Crystal Structures of the Copper-Containing Amine Oxidase from Arthrobacter globiformis in the Holo and Apo Forms:  Implications for the Biogenesis of Topaquinone^,

Abstract

The crystal structures of the copper enzyme phenylethylamine oxidase from the Gram-positive bacterium Arthrobacter globiformis (AGAO) have been determined and refined for three forms of the enzyme: the holoenzyme in its active form (at 2.2 A resolution), the holoenzyme in an inactive form (at 2.8 A resolution), and the apoenzyme (at 2.2 A resolution). The holoenzyme has a topaquinone (TPQ) cofactor formed from the apoenzyme by the post-translational modification of a tyrosine residue in the presence of Cu2+. Significant differences between the three forms of AGAO are limited to the active site. The polypeptide fold is closely similar to those of the amine oxidases from Escherichia coli [Parsons, M. R., et al. (1995) Structure 3, 1171-1184] and pea seedlings [Kumar, V., et al. (1996) Structure 4, 943-955]. In the active form of holo-AGAO, the active-site Cu atom is coordinated by three His residues and two water molecules in an approximately square-pyramidal arrangement. In the inactive form, the Cu atom is coordinated by the same three His residues and by the phenolic oxygen of the TPQ, the geometry being quasi-trigonal-pyramidal. There is evidence of disorder in the crystals of both forms of holo-AGAO. As a result, only the position of the aromatic group of the TPQ cofactor, but not its orientation about the Cbeta-Cgamma bond, is determined unequivocally. In apo-AGAO, electron density consistent with an unmodified Tyr occurs at a position close to that of the TPQ in the inactive holo-AGAO. This observation has implications for the biogenesis of TPQ. Two features which have not been described previously in amine oxidase structures are a channel from the molecular surface to the active site and a solvent-filled cavity at the major interface between the two subunits of the dimer.

Reversible inhibition of copper amine oxidase activity by channel-blocking ruthenium(II) and rhenium(I) molecular wires

“…The interaction may be either electrostatic or hydrophobic or both. In AGAO, the molecular surface surrounding the entrance to the active-site channel is predominantly negative (25). A wire with a Ru(II) head group (charge ϩ2) should bind better than one with a Re(I) head group (charge ϩ1) or a wire with no head group, as observed.…”

“…There is no deviation from the crystallographic twofold symmetry of the molecule, and the present crystallographic data do not permit us to conclude whether the two channels are occupied symmetrically. Within the limits of precision, the structure of the protein in the wire complex is identical to the structure of the native protein (25). The TPQ ring, which is disordered in many CuAO structures and is bound to the Cu atom in some of them, is fully ordered in the off-Cu position.…”

“…Other recent results also suggest that it may be feasible to design selective mechanism-based CuAO inhibitors. In the crystal structure of AGAO, the active-site channel and pocket are lined by 13 hydrophobic residues (25). When an inhibitor, 4-(2-naphthyloxy)-2-butyn-1-amine, was modeled into the AGAO active site as a Schiff base derivative, it made significant contacts with 5 of these residues (Phe-105, Trp-168, Tyr-302, Tyr-307, and Trp-359), and 37 Ϯ 3 nM (5b) [Ru(II)phen-C 43 Ϯ 1.5 nM (5c) [Ru(II)phen-C 80 Ϯ 11 nM (5d) [Ru(II)phen-C 92 Ϯ 9 nM (5e) [Ru(II)phen-C 92 Ϯ 11 nM (8) [Ru(II)phen-C 680 Ϯ 35 nM (10) [Re(I)phen-C 195 Ϯ 8 nM…”

A model for the reaction mechanism of the transglutaminase 3 enzyme

“…Crystal structures of des (1-52) grancalcin revealed that Ca 2+ -dependent conformational change is very small, and it is thought that the N-terminal Gly/Pro-rich region is required for the conformational change. A second example, copper enzyme phenylethylamine oxidase (PDB 1AV4), also shows a drastic reduction of B factors in almost all of its residues upon Cu 2+ and topaquinone cofactor binding (Wilce et al, 1997). The structures of the apo-and holoenzymes used for comparison have both been determined at 2.2 Å resolution, and the overall rms difference between them is 0.4 Å These examples indicate that local ligand binding can change the B factors of the residues distant from the binding sites.…”

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.…”