Identification of the Catalytic Base for Alcohol Activation in Choline Oxidase

Abstract

Choline oxidase catalyzes the oxidation of choline to glycine betaine through a two-step, four-electron reaction with betaine aldehyde as an intermediate. Oxygen is the final electron acceptor. Alcohol oxidation is initiated by the removal of the substrate hydroxyl proton by an unknown active site residue with a pKa value of ∼7.5. In the crystal structure of the enzyme in complex with glycine betaine, H466 is ≤3.1 Å from the carboxylate oxygen of the reaction product, suggesting a possible role in the proton abstraction reaction catalyzed by the enzyme. H466, along with another potential candidate, H351, was previously mutated to alanine, but this failed to establish if either residue was involved in activation of the substrate. In this study, single variants of choline oxidase with H466 and H351 substituted with glutamine were prepared, purified, and characterized. The kcat and kcat/Km values of the H351Q enzyme in atmospheric oxygen were 45- and 5000-fold lower than those of the wild-type enzyme, respectively, whereas the H466Q enzyme was inactive when assayed polarographically with choline. In the H466Q enzyme, the rate constant for anaerobic flavin reduction (kred) with choline was 1 million-fold lower than in the wild-type enzyme. A comparison of the fluorescence, circular dichroism, and (1)H nuclear magnetic resonance spectroscopic properties of the H466Q enzyme and the wild-type enzyme is consistent with the mutation not affecting the topology of the active site or the overall fold of the protein. Thus, the change in the kred value and the lack of oxygen consumption upon mutation of histidine to glutamine are not due to misfolded protein but rather to the variant enzyme being unable to catalyze substrate oxidation. On the basis of the kinetic and spectroscopic results presented here and the recent structural information, we propose that H466 is the residue that activates choline to the alkoxide for the subsequent hydride transfer reaction to the enzyme-bound flavin.

Crystal Structure of Alcohol Oxidase from Pichia pastoris

“…For other alcohol oxidases (AAO, GO, CDH, P2O, PNO) the conserved His of the His/His, His/Asn or His/Pro pairs is thought to act as catalytic base and thus carrying a partial positive charge during catalysis. Recently His466 of the closely related CO was identified as the catalytic base for the activation of the alcohol [ 53 ], which corresponds to His567 in AOX1 ( Fig 4C ). Suggested stepwise reaction catalyzed by AOX1 requires stabilization of the negative charge of the alkoxide by the enzyme through intermolecular interactions, which could be formed by two conserved polar residues, namely His567 (CO His466) and Asn616 (CO Asn 510), placed within a hydrogen bond distance to the proposed alkoxide position.…”

Crystallographic fragment screening-based study of a novel FAD-dependent oxidoreductase from Chaetomium thermophilum

“…The structural, mechanistic and computational studies show that the conserved active-site histidine is likely to play the role of the catalytic base deprotonating the hydroxyl group of a substrate in AnGOX, PeAAO, HMFO and other GMC enzymes (Herná ndez-Ortega et al, 2012;Wongnate & Chaiyen, 2013;Smitherman et al, 2015;Leskovac et al, 2005;Mugo et al, 2013;Graf et al, 2015;Sygmund et al, 2013;Dijkman et al, 2015). In CtFDO, the position of the active-site histidine is occupied by His564 (Fig.…”

“…2). The position of His564 in CtFDO corresponds to the conserved active-site histidine residue that plays the role of a catalytic base during the reductive half-reaction in the majority of GMC oxidoreductases (Herná ndez-Ortega et al, 2012; Wongnate & Chaiyen, 2013;Smitherman et al, 2015;Leskovac et al, 2005;Graf et al, 2015;Mugo et al, 2013;Sygmund et al, 2013). The orientation of the His564 imidazole ring is stabilized by a hydrogen bond to Gln351 (His564 N 1 -Gln351 O "1 ).…”

“…Although the GMC oxidoreductases catalyse the oxidation of different types of substrates, they share core structural elements, a two-domain character, a conserved N-terminal Gly-X-Gly-X-X-Gly sequence motif characteristic of the initial segment of the Rossmann fold that binds the adenosine diphosphate moiety of FAD, and a conserved active-site histidine. The conserved histidine plays the role of the catalytic base in the majority of GMC oxidoreductases (Romero & Gadda, 2014;Wohlfahrt et al, 1999;Herná ndez-Ortega et al, 2012;Yoshida et al, 2015;Wongnate & Chaiyen, 2013;Mugo et al, 2013;Graf et al, 2015;Smitherman et al, 2015;Dijkman et al, 2015) and is usually present in combination with another histidine or an asparagine residue, which can form a hydrogen bond to an alcohol substrate. Both residues are situated on the re face of the FAD isoalloxazine ring, creating a His-His or His-Asn pair (Sü tzl et al, 2019).…”

Comparative Computational Approach To Study Enzyme Reactions Using QM and QM-MM Methods

“…However, the resolution of the crystal structure of choline oxidase with the glycine betaine 20 as the enzyme–product complex (PC) led to the revisiting of the concept of His351 and His466 as the possible active-site bases for the deprotonation of O–H as a result of the close interaction of these residues with the glycine betaine. 15 Genetic modification of His466 with glutamine led to the conclusion that His466 is the probable catalytic base. A plausible scenario was offered to explain why alanine variant could not verify the role of His466 in the catalysis process.…”