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

Abstract

Background:Copper amine oxidases catalyze amine oxidation using copper and a quinone cofactor. Results: Halides bind axially to the copper center, preventing the reduced cofactor from adopting an on-copper conformation. Conclusion:The cofactor undergoes large conformational changes during the catalytic reaction that enable transitions between different types of chemistry. Significance: Molecular details of cofactor movement have been unveiled based on structural and kinetic evidence.

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

“…7 and 8 in ref. 8), those determined at four different noncryogenic temperatures clearly showed a mixture of the two conformers (off-copper TPQ amr and on-copper TPQ sq ) in the F o -F c omit maps for residue 382 (TPQ; Fig. 2A).…”

“…Our previous study with AGAO crystals anaerobically reduced with a high-affinity substrate, 2phenylethylamine (2-PEA; K m = 2.5 μM) (4, 6), revealed that the oxidized product, phenylacetaldehyde (PAA), remains bound in the hydrophobic pocket close to TPQ, which could affect the TPQ sq /TPQ amr equilibrium (8). Therefore, we here used a lowaffinity substrate, ethylamine (K m = 170 mM) (6), as a reducing substrate, expecting that its oxidation product (acetaldehyde) is not bound to the pocket (8), and hence would not affect the TPQ sq /TPQ amr equilibrium to be investigated by X-ray crystallography, using the temperature-controlled HAG method. Diffraction data were obtained with multiple crystals selected arbitrarily from those formed in the same soaking conditions except for the temperature (4, 10, 15, and 20°C) (SI Appendix, Table S1).…”

“…1B) (8). Previous spectrophotometric studies revealed that the equilibrium between TPQ sq and TPQ amr is affected significantly by temperature and pH (8,9); at higher pH values and temperatures, the equilibrium shifts toward TPQ sq , which suggests that the conformational change of TPQ is accompanied by a change in enthalpy. However, the structural basis for the pH-and temperaturedependent equilibrium remains unresolved.…”

“…All CuAOs so far characterized structurally are the homodimers of a 70-95-kDa subunit, each containing a Cu 2+ ion and a protein-derived quinone cofactor, topaquinone (TPQ) (3). We have determined X-ray crystal structures of the enzyme from Arthrobacter globiformis (AGAO) that had been reduced with substrate under anaerobic conditions (4)(5)(6)(7)(8) and demonstrated that the reduced cofactor formed in the initial reductive half-reaction has two distinct conformations: a semiquinone radical form (TPQ sq ), in which the 4-hydroxyl group of the cofactor is ligated axially to the copper center, and an aminoresorcinol form (TPQ amr ), in which the cofactor has no direct contact with the copper (designated "oncopper" and "off-copper" conformations, respectively; Fig. 1B) (8).…”

“…We have determined X-ray crystal structures of the enzyme from Arthrobacter globiformis (AGAO) that had been reduced with substrate under anaerobic conditions (4)(5)(6)(7)(8) and demonstrated that the reduced cofactor formed in the initial reductive half-reaction has two distinct conformations: a semiquinone radical form (TPQ sq ), in which the 4-hydroxyl group of the cofactor is ligated axially to the copper center, and an aminoresorcinol form (TPQ amr ), in which the cofactor has no direct contact with the copper (designated "oncopper" and "off-copper" conformations, respectively; Fig. 1B) (8). Previous spectrophotometric studies revealed that the equilibrium between TPQ sq and TPQ amr is affected significantly by temperature and pH (8,9); at higher pH values and temperatures, the equilibrium shifts toward TPQ sq , which suggests that the conformational change of TPQ is accompanied by a change in enthalpy.…”