Development of Direct Electron Transfer-Type Cascade System by Alcohol and Aldehyde Dehydrogenases

概要

Oxidoreductases are biocatalysts with high activity and substrate specificity under mild
conditions (room temperature, normal pressure, and neutral pH), which realize efficient energy
conversions for a sustainable low-carbon society. Some redox enzymes can proceed with catalytic
reactions on electrically-connected electrode materials, which is called direct electron transfer
(DET)-type bioelectrocatalysis (Fig. 1). DET-type reactions are expected to be applied to
bioelectrochemical devices such as biosensors, biofuel cells, and bioreactors.
In this study, we focused on membrane-bound alcohol dehydrogenase (ADH) and
aldehyde dehydrogenase (AlDH) from Gluconobacter oxydans. They can catalyze two-step
oxidation from ethanol to acetate via acetaldehyde in the respiratory electron transfer system called
acetic acid fermentation. Both enzymes are heterotrimers with the catalytic centers of
pyrroloquinoline quinone (PQQ) for ADH and molybdopterin (Moco) for AlDH, respectively. The
electron transfer pathways are estimated to be from the catalytic centers where substrates are
oxidized to the final natural electron acceptor, ubiquinone-10 (UQ10), via the membrane-bound
cytochrome c subunits composed of three hemes c (called hemes 1c, 2c, and 3c from the N-terminus
in this order). However, since the structures of both enzymes are still unknown, the catalytic
mechanism has not been elucidated.
On the other hand, the DET activity of ADH was confirmed in a previous report, but
details such as the suitability of electrode materials and the electron transfer mechanism are not clear.
In addition, no electrochemical studies on ALDH have been conducted. In this study, we attempted
the basic evaluation of DET-type reaction of ADH and AlDH from the viewpoint of structural
biology and bioelectrochemistry, and its application development.
In Chapter 1, the author attempted to investigate characteristics of ADH and AlDH from
viewpoints of structural biology and bioelectrochemistry. First, three-dimensional (3D) structures of
ADH and AlDH were elucidated using cryo-electron microscopy (cryo-EM) and single particle
image analysis. DET-type bioelectrocatalysis by the two enzymes was then characterized based on
their 3D structures. Particularly, the author focused on the cyanide (CN–) effects on their
bioelectrocatalytic properties, because CN– coordination to hemes c was expected to interfere with
the heme c-related electron transfer pathways of ADH and AlDH. Their bioelectrochemical
characteristics were compared based on the CN– effects from viewpoints of kinetics and
thermodynamics.
In Chapter 2, the author attempted to develop bienzymatic DET-type cascade system with
ADH and AlDH. First, DET-type bioelectrocatalysis by ADH and AlDH was improved at
pyrene-derivative-functionalized multi-walled carbon nanotubes. ...