High-resolution crystal structure of copper amine oxidase fromArthrobacter globiformis: assignment of bound diatomic molecules as O₂

Abstract

The crystal structure of a copper amine oxidase from Arthrobacter globiformis was determined at 1.08 Å resolution with the use of low-molecular-weight polyethylene glycol (LMW PEG; average molecular weight ∼200) as a cryoprotectant. The final crystallographic R factor and Rfree were 13.0 and 15.0%, respectively. Several molecules of LMW PEG were found to occupy cavities in the protein interior, including the active site, which resulted in a marked reduction in the overall B factor and consequently led to a subatomic resolution structure for a relatively large protein with a monomer molecular weight of ∼70,000. About 40% of the presumed H atoms were observed as clear electron densities in the Fo - Fc difference map. Multiple minor conformers were also identified for many residues. Anisotropic displacement fluctuations were evaluated in the active site, which contains a post-translationally derived quinone cofactor and a Cu atom. Furthermore, diatomic molecules, most likely to be molecular oxygen, are bound to the protein, one of which is located in a region that had previously been proposed as an entry route for the dioxygen substrate from the central cavity of the dimer interface to the active site.

In crystallo thermodynamic analysis of conformational change of the topaquinone cofactor in bacterial copper amine oxidase

“…Materials. Recombinant AGAO was purified as its inactive precursor form and converted to the Cu/TPQ-containing active form, as reported previously (19,20). Protein and TPQ sq concentrations were determined spectrophotometrically, using molar extinction coefficients of e 280 = 93,200 M −1 •cm −1 (19) and e 450 = 5,000 M −1 •cm −1 (12), respectively.…”

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

“…9). Met-602 is located at the end of the predicted O 2 pathway from the O 2 -prebinding site to the copper center and has conformational flexibility with dual extreme conformers (19). Thus, it is tempting to speculate that the tethering of the Met-602 side chain could act as a gate to allow O 2 to enter into the copper center in the initial phase of the oxidative half-reaction.…”

“…Materials-Recombinant AGAO was purified as its inactive precursor and converted to the copper-and TPQ-containing active form as reported previously (12,19). Protein and TPQ sq concentrations were determined spectrophotometrically using molar extinction coefficients of ⑀ 280 ϭ 93,200 M Ϫ1 cm Ϫ1 (12) and ⑀ 468 ϭ 4500 M Ϫ1 cm Ϫ1 , respectively (35).…”

“…As summarized in a recent review (3), x-ray crystal structures of CAOs have been determined for a number of enzymes from various sources, including bacteria (Escherichia coli (16) and Arthrobacter globiformis (AGAO) (17)(18)(19)), yeasts (Hansenula polymorpha (recently reclassified as Pichia angusta) (HPAO-1 and HPAO-2) (20,21) and Pichia pastoris (lysyl oxidase) (22)), a fungus (Aspergillus nidulans (23)), a plant (Pisum savitum (24)), a mammal (Bos taurus (25)), and Homo sapiens (diamine oxidase and vascular adhesion protein 1, which is identical to semicarbazide-sensitive amine oxidase (26 -28)). The active site structures of these enzymes, including the positions of TPQ and Cu(II), are highly conserved, suggesting that they have a common mechanism for single-turnover TPQ biogenesis and catalytic amine oxidation.…”

Investigation of dioxygen activation by copper(ii)–iminate/aminate complexes

“…17,[48][49][50][51] The electron transfer from aminotopaquinol to dioxygen results in the formation of superoxide which oxidizes imino-topaquinone (Scheme 1B). 17,50,[52][53][54] Anice example of copper enzyme with a - 2 : 2 -peroxide mode of ligation is the recently discovered binuclear copper enzyme NspF, which activates dioxygen to convert 3-amino-4-hydroxybenzamide to 4-hydroxy-3-nitrosobenzamide (Scheme 1B). 55 The NspFenzyme, like in other copper monoxygenases, reacts with dioxygen to form a - 2 : 2 -peroxide adduct.…”