1 3
Topics in Current Chemistry (2018) 376:43
coworkers demonstrated the photoelectrochemical interpretation of a catalytic cascade originally reported by Obert and Bakul [35] employing a three-enzyme cascade
including FDH, formaldehyde dehydrogenase (FaldDH), and ADH in the reverse
directions from that described above to reduce CO 2 to formate, formaldehyde and
then methanol [36]. Simultaneously, NADH was regenerated from NAD
+
via a nonenzymatic cascade in which photoexcited electrons are transferred through a BiFeO 3
photocathode to a rhodium-based electrochemical mediator ([Cp*Rh(bpy)H 2 O]
+
,
Cp* = C 5 Me 5 , bpy = 2,2′-bipyridine). Coupled with a Co–P i /α–Fe 2 O 3 photoanode,
this enzymatic cascade was able to reduce CO 2 to methanol in a divided photoelectrochemical cell under mild aqueous conditions (pH 7, 25 °C). This work illustrates
the intricate level of complexity that can be achieved in designing hybrid electrometabolic pathways; however, expanding research of highly complex uncompartmentalized catalytic cascades requires strategies to mitigate diffusional and kinetic
constraints.
6 Spatial Orientation and Substrate Channeling
As discussed above, the primary issues plaguing design of electrometabolic pathways arises from the multiplicity of intermediates and cofactors and their uncontrolled diffusion throughout the bulk media. As artificial pathways increase in complexity, there is an increasing probability that mismatched enzyme kinetics will lead
to a buildup of intermediates. This reactivity bottleneck will in turn lead to product inhibition and ultimately result in sluggish cascade rates. Nature has evolved to
overcome this problem through a combination of compartmentalization and spatial
orientation of enzymes. A growing field of research has focused on fundamentally
understanding the role that spatial organization plays in naturally occurring pathways and exploiting that knowledge in the design of artificial reaction cascades.
Enzymes that react sequentially can form transient non-covalent complexes,
termed metabolons, that enhance metabolic flux by minimizing the distance required
for a common reaction intermediate to diffuse from one enzyme to the next. Several groups have employed a strategy of co-immobilization of sequentially reacting
enzymes onto a well-defined molecular scaffold to enable precise control over the
ordering, ratio, and diffusional length scale between enzymes. Multiple synthetic
and biologically engineered scaffolds have been employed, ranging from virus-based
nanocages [37] to hydrophobized silica macropores [38] to DNA origami [39]. One
scaffold that we will highlight here utilizes a series of selective binding proteins that
natively exist to help form the cellulase complex (known as the cellulosome). These
binding protein couples consist of two units, cohesins and dockerins, that exhibit a
high binding specificity for one another; this specificity was exploited by Chen and
coworkers (among others [40]) to spatially orient three sequential dehydrogenases
(alcohol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase)
to catalyze the oxidation of methanol to CO 2 [41]. Each enzyme was genetically
engineered to contain a specific dockerin tag at the C-termini, that bind to the corresponding cohesin domain of a miniscaffoldin displayed on a yeast cell surface.
The authors demonstrated that the scaffoldin-bound enzyme sequence exhibited a
Reprinted from the journal
159
Topics in Current Chemistry (2018) 376:43
coworkers demonstrated the photoelectrochemical interpretation of a catalytic cascade originally reported by Obert and Bakul [35] employing a three-enzyme cascade
including FDH, formaldehyde dehydrogenase (FaldDH), and ADH in the reverse
directions from that described above to reduce CO 2 to formate, formaldehyde and
then methanol [36]. Simultaneously, NADH was regenerated from NAD
+
via a nonenzymatic cascade in which photoexcited electrons are transferred through a BiFeO 3
photocathode to a rhodium-based electrochemical mediator ([Cp*Rh(bpy)H 2 O]
+
,
Cp* = C 5 Me 5 , bpy = 2,2′-bipyridine). Coupled with a Co–P i /α–Fe 2 O 3 photoanode,
this enzymatic cascade was able to reduce CO 2 to methanol in a divided photoelectrochemical cell under mild aqueous conditions (pH 7, 25 °C). This work illustrates
the intricate level of complexity that can be achieved in designing hybrid electrometabolic pathways; however, expanding research of highly complex uncompartmentalized catalytic cascades requires strategies to mitigate diffusional and kinetic
constraints.
6 Spatial Orientation and Substrate Channeling
As discussed above, the primary issues plaguing design of electrometabolic pathways arises from the multiplicity of intermediates and cofactors and their uncontrolled diffusion throughout the bulk media. As artificial pathways increase in complexity, there is an increasing probability that mismatched enzyme kinetics will lead
to a buildup of intermediates. This reactivity bottleneck will in turn lead to product inhibition and ultimately result in sluggish cascade rates. Nature has evolved to
overcome this problem through a combination of compartmentalization and spatial
orientation of enzymes. A growing field of research has focused on fundamentally
understanding the role that spatial organization plays in naturally occurring pathways and exploiting that knowledge in the design of artificial reaction cascades.
Enzymes that react sequentially can form transient non-covalent complexes,
termed metabolons, that enhance metabolic flux by minimizing the distance required
for a common reaction intermediate to diffuse from one enzyme to the next. Several groups have employed a strategy of co-immobilization of sequentially reacting
enzymes onto a well-defined molecular scaffold to enable precise control over the
ordering, ratio, and diffusional length scale between enzymes. Multiple synthetic
and biologically engineered scaffolds have been employed, ranging from virus-based
nanocages [37] to hydrophobized silica macropores [38] to DNA origami [39]. One
scaffold that we will highlight here utilizes a series of selective binding proteins that
natively exist to help form the cellulase complex (known as the cellulosome). These
binding protein couples consist of two units, cohesins and dockerins, that exhibit a
high binding specificity for one another; this specificity was exploited by Chen and
coworkers (among others [40]) to spatially orient three sequential dehydrogenases
(alcohol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase)
to catalyze the oxidation of methanol to CO 2 [41]. Each enzyme was genetically
engineered to contain a specific dockerin tag at the C-termini, that bind to the corresponding cohesin domain of a miniscaffoldin displayed on a yeast cell surface.
The authors demonstrated that the scaffoldin-bound enzyme sequence exhibited a
Reprinted from the journal
159