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Oxygen activation switch in the copper amine oxidase of Escherichia coli

DOI: 10.1021/acs.biochem.8b00633 DOI Help

Authors: Thembaninkosi G. Gaule (University of Leeds) , Mark A. Smith (University of Leeds) , Katarzyna M. Tych (University of Leeds; Technische Universität München) , Pascale Pirrat (University of Leeds) , Chi H. Trinh (University of Leeds) , Arwen R. Pearson (University of Leeds; Universität Hamburg) , Peter F. Knowles (University of Leeds) , Michael J. Mcpherson (University of Leeds)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemistry

State: Published (Approved)
Published: August 2018
Diamond Proposal Number(s): 6386

Abstract: Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu2+ in the active site, two peripheral calcium sites, ca. 32 Å from the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Ca2+ (Asp533, Leu534, Asp535, Asp678, Ala 679) is essential for full-length protein production, while the surface Ca2+ (Glu573, Tyr667, Asp670, Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The mutation E573Q at the surface-site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis (ca. 60 % WTECAO TPQ), I342F/E573Q has almost no amine oxidase activity (ca. 4.6 % WTECAO activity). To understand how these long range mutations result in major effects on TPQ biogenesis and catalysis we employed UV-vis spectroscopy, steady-state kinetics, inhibition assays and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu2+-substrate reduced TPQ (TPQAMQ) Cu1+-TPQ semiquinone (TPQSQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to the Cu2+-TPQAMQ or destabilizes the Cu1+-TPQSQ. Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu2+-tyrosinate state. Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and whilst a Cu1+-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis.

Subject Areas: Chemistry, Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography