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Powering Artificial Enzymatic Cascades with Electrical Energy.

ABSTRACT: We have developed a scalable platform that employs electrolysis for an in?vitro synthetic enzymatic cascade in a continuous flow reactor. Both H2 and O2 were produced by electrolysis and transferred through a gas-permeable membrane into the flow system. The membrane enabled the separation of the electrolyte from the biocatalysts in the flow system, where H2 and O2 served as electron mediators for the biocatalysts. We demonstrate the production of methylated N-heterocycles from diamines with up to 99?% product formation as well as excellent regioselective labeling with stable isotopes. Our platform can be applied for a broad panel of oxidoreductases to exploit electrical energy for the synthesis of fine chemicals.

SUBMITTER: Al-Shameri A

PROVIDER: S-EPMC7318245 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Powering Artificial Enzymatic Cascades with Electrical Energy.

Al-Shameri Ammar A Petrich Marie-Christine MC Junge Puring Kai K Apfel Ulf-Peter UP Nestl Bettina M BM Lauterbach Lars L

Angewandte Chemie (International ed. in English) 20200428 27

We have developed a scalable platform that employs electrolysis for an in vitro synthetic enzymatic cascade in a continuous flow reactor. Both H2 and O2 were produced by electrolysis and transferred through a gas-permeable membrane into the flow system. The membrane enabled the separation of the electrolyte from the biocatalysts in the flow system, where H2 and O2 served as electron mediators for the biocatalysts. We demonstrate the production of methy ...[more]

PMID: 32202370

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Project description:Artificial metalloenzymes (ArMs) result from anchoring a metal-containing moiety within a macromolecular scaffold (protein or oligonucleotide). The resulting hybrid catalyst combines attractive features of both homogeneous catalysts and enzymes. This strategy includes the possibility of optimizing the reaction by both chemical (catalyst design) and genetic means leading to achievement of a novel degree of (enantio)selectivity, broadening of the substrate scope, or increased activity, among others. In the past 20 years, the Ward group has exploited, among others, the biotin-(strept)avidin technology to localize a catalytic moiety within a well-defined protein environment. Streptavidin has proven versatile for the implementation of ArMs as it offers the following features: (i) it is an extremely robust protein scaffold, amenable to extensive genetic manipulation and mishandling, (ii) it can be expressed in E. coli to very high titers (up to >8 g·L-1 in fed-batch cultures), and (iii) the cavity surrounding the biotinylated cofactor is commensurate with the size of a typical metal-catalyzed transition state. Relying on a chemogenetic optimization strategy, varying the orientation and the nature of the biotinylated cofactor within genetically engineered streptavidin, 12 reactions have been reported by the Ward group thus far. Recent efforts within our group have focused on extending the ArM technology to create complex systems for integration into biological cascade reactions and in vivo. With the long-term goal of complementing in vivo natural enzymes with ArMs, we summarize herein three complementary research lines: (i) With the aim of mimicking complex cross-regulation mechanisms prevalent in metabolism, we have engineered enzyme cascades, including cross-regulated reactions, that rely on ArMs. These efforts highlight the remarkable (bio)compatibility and complementarity of ArMs with natural enzymes. (ii) Additionally, multiple-turnover catalysis in the cytoplasm of aerobic organisms was achieved with ArMs that are compatible with a glutathione-rich environment. This feat is demonstrated in HEK-293T cells that are engineered with a gene switch that is upregulated by an ArM equipped with a cell-penetrating module. (iii) Finally, ArMs offer the fascinating prospect of "endowing organometallic chemistry with a genetic memory." With this goal in mind, we have identified E. coli's periplasmic space and surface display to compartmentalize an ArM, while maintaining the critical phenotype-genotype linkage. This strategy offers a straightforward means to optimize by directed evolution the catalytic performance of ArMs. Five reactions have been optimized following these compartmentalization strategies: ruthenium-catalyzed olefin metathesis, ruthenium-catalyzed deallylation, iridium-catalyzed transfer hydrogenation, dirhodium-catalyzed cyclopropanation and carbene insertion in C-H bonds. Importantly, >100 turnovers were achieved with ArMs in E. coli whole cells, highlighting the multiple turnover catalytic nature of these systems.

Project description:A fundamental re-assessment of the overall energetics of biochemical electron transfer chains and cycles is presented, highlighting the crucial role of the highest-energy molecule involved, O2. The chemical energy utilized by most complex multicellular organisms is not predominantly stored in glucose or fat, but rather in O2 with its relatively weak (i.e., high-energy) double bond. Accordingly, reactions of O2 with organic molecules are highly exergonic, while other reactions of glucose, fat, NAD(P)H, or ubiquinol (QH2) are not, as demonstrated in anaerobic respiration with its meager energy output. The notion that "reduced molecules" such as alkanes or fatty acids are energy-rich is shown to be incorrect; they only unlock the energy of more O2, compared to O-containing molecules of similar mass. Glucose contains a moderate amount of chemical energy per bond (<20% compared to O2), as confirmed by the relatively small energy output in glycolysis and the Krebs cycle converting glucose to CO2 and NADH. Only in the "terminal" aerobic respiration reaction with O2 does a large free energy change occur due to the release of oxygen's stored chemical energy. The actual reaction of O2 in complex IV of the inner mitochondrial membrane does not even involve any organic fuel molecule and yet releases >1 MJ when 6 mol of O2 reacts. The traditional presentation that relegated O2 to the role of a low-energy terminal acceptor for depleted electrons has not explained these salient observations and must be abandoned. Its central notion that electrons release energy because they move from a high-energy donor to a low-energy acceptor is demonstrably false. The energies of (at least) two donor and two acceptor species come into play, and the low "terminal" negative reduction potential in aerobic respiration can be attributed to the unusually high energy of O2, the crucial reactant. This is confirmed by comparison with the corresponding half-reaction without O2, which is endergonic. In addition, the electrons are mostly not accepted by oxygen but by hydrogen. Redox energy transfer and release diagrams are introduced to provide a superior representation of the energetics of the various species in coupled half-reactions. Electron transport by movement of reduced molecules in the electron transfer chain is shown to run counter to the energy flow, which is carried by oxidized species. O2, rather than glucose, NAD(P)H, or ATP, is the molecule that provides the most energy to animals and plants and is crucial for sustaining large complex life forms. The analysis also highlights a significant discrepancy in the proposed energetics of reactions of aerobic respiration, which should be re-evaluated.

Dataset Information

Powering Artificial Enzymatic Cascades with Electrical Energy.

Publications

Powering Artificial Enzymatic Cascades with Electrical Energy.

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