3LJP

Crystal structure of choline oxidase V464A mutant

Summary for 3LJP

• Entry DOI: 10.2210/pdb3ljp/pdb
• Descriptor: Choline oxidase, DIHYDROFLAVINE-ADENINE DINUCLEOTIDE (3 entities in total)
• Functional Keywords: flavoenzyme oxidase, covalently linked fad, fad, flavoprotein, oxidoreductase
• Biological source: Arthrobacter globiformis
• Total number of polymer chains: 2
• Total formula weight: 121316.55

Authors

Finnegan, S.,Agniswamy, J.,Weber, I.T.,Giovanni, G. (deposition date: 2010-01-26, release date: 2010-03-16, Last modification date: 2023-09-06)

Primary citation

Finnegan, S.,Agniswamy, J.,Weber, I.T.,Gadda, G.
Role of valine 464 in the flavin oxidation reaction catalyzed by choline oxidase.
Biochemistry, 49:2952-2961, 2010

PubMed Abstract: The oxidation of reduced flavin cofactors by oxygen is a very important reaction that is central to the chemical versatility of hundreds of flavoproteins classified as monooxygenases and oxidases. These enzymes are characterized by bimolecular rate constants >or=10(5) M(-1) s(-1) and produce water and hydrogen peroxide, respectively. A hydrophobic cavity close to the reactive flavin C(4a) atom has been previously identified in the 3D structure of monooxygenases but not in flavoprotein oxidases. In the present study, we have investigated by X-ray crystallography, mutagenesis, steady-state, and rapid reaction approaches the role of Val464, which is <6 A from the flavin C(4a) atom in choline oxidase. The 3D structure of the Val464Ala enzyme was essentially identical to that of the wild-type enzyme as shown by X-ray crystallography. Time-resolved anaerobic substrate reduction of the enzymes showed that replacement of Val464 with alanine or threonine did not affect the reductive half-reaction. Steady-state and rapid kinetics as well as enzyme-monitored turnovers indicated that the oxidative half-reaction in the Ala464 and Thr464 enzymes was decreased by approximately 50-fold with respect to the wild-type enzyme. We propose that the side chain of Val464 in choline oxidase provides a nonpolar site that is required to guide oxygen in proximity of the C(4a) atom of the flavin, where it will subsequently react via electrostatic catalysis. Visual analysis of available structures suggests that analogous nonpolar sites are likely present in most flavoprotein oxidases. Mechanistic considerations provide rationalization for the differences between sites in monooxygenases and oxidases.

PubMed: 20184377
DOI: 10.1021/bi902048c

Experimental method

X-RAY DIFFRACTION (2.2 Å)