Alteration of Electron Acceptor Preferences in the Oxidative Half-Reaction of Flavin-Dependent Oxidases and Dehydrogenases

Abstract

In this review, recent progress in the engineering of the oxidative half-reaction of flavin-dependent oxidases and dehydrogenases is discussed, considering their current and future applications in bioelectrochemical studies, such as for the development of biosensors and biofuel cells. There have been two approaches in the studies of oxidative half-reaction: engineering of the oxidative half-reaction with oxygen, and engineering of the preference for artificial electron acceptors. The challenges for engineering oxidative half-reactions with oxygen are further categorized into the following approaches: (1) mutation to the putative residues that compose the cavity where oxygen may be located, (2) investigation of the vicinities where the reaction with oxygen may take place, and (3) investigation of possible oxygen access routes to the isoalloxazine ring. Among these approaches, introducing a mutation at the oxygen access route to the isoalloxazine ring represents the most versatile and effective strategy. Studies to engineer the preference of artificial electron acceptors are categorized into three different approaches: (1) engineering of the charge at the residues around the substrate entrance, (2) engineering of a cavity in the vicinity of flavin, and (3) decreasing the glycosylation degree of enzymes. Among these approaches, altering the charge in the vicinity where the electron acceptor may be accessed will be most relevant.

Questions in the Chemical Enzymology of MAO

“…For electrode use, an alternative to oxygen as the electron acceptor in the oxidative half-reaction is desirable. Methylene Blue and similar dyes are known acceptors [88,89], but performance could be improved by rational engineering of the oxygen pathway or reaction site [90]. This has been demonstrated for MAO A by mutating Lys305: after reduction by serotonin, the K305M MAO A reoxidation by oxygen was much slower but electrons could pass instead to a quinone [91].…”

Structure of lactate oxidase from Enterococcus hirae revealed new aspects of active site loop function: Product‐inhibition mechanism and oxygen gatekeeper

“…The reaction mechanism of oxygen in flavin‐dependent oxidases were investigated in many kinds of enzymes but is still not fully elucidated. However, the key to the alteration of the reactivity for oxygen can be divided into three methods by accumulated literature: (a) mutation to the putative residues that compose the cavity where oxygen is located, (b) mutation into the vicinities where the reaction with oxygen takes place, and (c) mutation around possible oxygen‐access routes to the isoalloxazine ring 48 . EhLOx Met207 is far from FMN isoalloxazine C4a, where is the reaction site with molecular oxygen (distance between Cβ and FMN C4a is 10.8 Å in state A and 10.3 Å in state B), or the corresponding oxygen binding site of AvLOx 49 .…”

“…(a) mutation to the putative residues that compose the cavity where oxygen is located, (b) mutation into the vicinities where the reaction with oxygen takes place, and (c) mutation around possible oxygen-access routes to the isoalloxazine ring. 48 EhLOx Met207 is far from FMN isoalloxazine C4a, where is the reaction site with molecular oxygen (distance between Cβ and FMN C4a is 10.8 Å in state A and 10.3 Å in state B), or the corresponding oxygen binding site of AvLOx. 49 Many flavin-dependent oxidases and dehydrogenases had their reactivity for oxygen altered by the second method, mutation into the vicinities of flavin, which is in 3.7-7.3 Å from isoalloxazine C4a.…”

“…Many flavin-dependent oxidase studies have already revealed the availability of the prediction of an oxygen-accessible channel. 48 The software CAVER was used for analysis, which is a powerful tool for tunnel analysis in proteins based on reciprocal distance function grid. 56 CAVER analysis was performed using the CAVER 3.0.3 plugin of PyMOL and only the minimum probe radius was changed to 0.95 Å from default value referred to in the previous report from Collazo and Klinman.…”

Role of phenazine‐enzyme physiology for current generation in a bioelectrochemical system

“…The PQQ‐dependent enzyme reaction is believed to use the same (hydrid) transfer mechanism for both alcohol (Edh) and aldehyde (Gcd) dehydrogenases (Anthony, 1996; Oubrie et al ., 1999). Nonetheless, factors such as differences in the charged amino acid residues around the substrate entrance pocket might still influence electron acceptor preference (Hiraka et al ., 2020). The mode of extracellular electron transfer achieved in vivo using phenazines or other redox mediators, however, does not only depend on the enzymes, which directly catalyse the reduction of these mediators.…”