skip to main content

Title: Preparation of Artificial Metalloenzymes
Transition metal catalysts and enzymes are ubiquitous tools for chemical synthesis. Potential benefits of combining complementary properties of these catalysts have driven efforts to create artificial metalloenzymes (ArMs), hybrid constructs comprised of synthetic metal centers embedded within protein scaffolds. This unique composition necessitates the use of synthetic chemistry, bioconjugation methodology, and protein engineering for ArM formation. Despite this challenge, a range of approaches for ArM formation have been developed. This review provides an overview these different approaches and discussion of potential advantages and disadvantages of each.
Authors:
;
Editors:
Diégues, M.; Bäckvall, J.-E.; Pàmies, O.
Award ID(s):
1839154
Publication Date:
NSF-PAR ID:
10317614
Journal Name:
Artificial Metalloenzymes and MetalloDNAzymes in Catalysis: From Design to Applications
Sponsoring Org:
National Science Foundation
More Like this
  1. Microorganisms are remarkable chemists, with enzymes as their tools for executing multi-step syntheses to yield myriad natural products. Microbial synthetic aptitudes are illustrated by the structurally diverse 2,5-diketopiperazine (DKP) family of bioactive nonribosomal peptide natural products. Nonribosomal peptide synthetases (NRPSs) have long been recognized as catalysts for formation of DKP scaffolds from two amino acid substrates. Cyclodipeptide synthases (CDPSs) are more recently recognized catalysts of DKP assembly, employing two aminoacyl-tRNAs (aa-tRNAs) as substrates. CDPS-encoding genes are typically found in genomic neighbourhoods with genes encoding additional biosynthetic enzymes. These include oxidoreductases, cytochrome P450s, prenyltransferases, methyltransferases, and cyclases, which equip the DKPmore »scaffold with groups that diversify chemical structures and confer biological activity. These tailoring enzymes have been characterized from nine CDPS-containing biosynthetic pathways to date, including four during the last year. In this review, we highlight these nine DKP pathways, emphasizing recently characterized tailoring reactions and connecting new developments to earlier findings. Featured pathways encompass a broad spectrum of chemistry, including the formation of challenging C–C and C–O bonds, regioselective methylation, a unique indole alkaloid DKP prenylation strategy, and unprecedented peptide-nucleobase bond formation. These CDPS-containing pathways also provide intriguing models of metabolic pathway evolution across related and divergent microorganisms, and open doors to synthetic biology approaches for generation of DKP combinatorial libraries. Further, bioinformatics analyses support that much unique genetically encoded DKP tailoring potential remains unexplored, suggesting opportunities for further expansion of Nature's biosynthetic spectrum. Together, recent studies of DKP pathways demonstrate the chemical ingenuity of microorganisms, highlight the wealth of unique enzymology provided by bacterial biosynthetic pathways, and suggest an abundance of untapped biosynthetic potential for future exploration.« less
  2. The primary and secondary coordination spheres of metal binding sites in metalloproteins have been investigated extensively, leading to the creation of high-performing functional metalloproteins; however, the impact of the overall structure of the protein scaffold on the unique properties of metalloproteins has rarely been studied. A primary example is the binuclear CuA center, an electron transfer cupredoxin domain of photosynthetic and respiratory complexes and, recently, a protein co-regulated with particulate methane and ammonia monooxygenases. The redox potential, Cu–Cu spectroscopic features, and a valence delocalized state of CuA are difficult to reproduce in synthetic models, and every artificial protein CuA centermore »to-date has used a modified cupredoxin. Here we present a fully functional CuA center designed in a structurally non-homologous protein, cytochrome c peroxidase (CcP), by only two mutations (CuACcP). We demonstrate with UV-visible absorption, resonance Raman, and MCD spectroscopy that CuACcP is valence delocalized. CW and pulsed (HYSCORE) X-band EPR show it has a highly compact gz area and small Az hyperfine principal value with g and A tensors that resemble axially perturbed CuA. Stopped-flow kinetics found that CuA formation proceeds through a single T2Cu intermediate. The reduction potential of CuACcP is comparable to native CuA and can transfer electrons to a physiological redox partner. We built a structural model of the designed Cu binding site from EXAFS and validated it by mutation of coordinating Cys and His residues, revealing that a triad of residues (R48C, W51C, and His52) rigidly arranged on one α-helix is responsible for chelating the first Cu atom and that His175 stabilizes the binuclear complex by rearrangement of the CcP heme-coordinating helix. This design is a demonstration that a highly conserved protein fold is not uniquely necessary to induce certain characteristic physical and chemical properties to a metal redox center.« less
  3. Hydration of nitriles is catalyzed by the enzyme nitrile hydratase, with iron or cobalt active sites, and by a variety of synthetic metal complexes. This Perspective focuses on parallels between the reaction mechanism of the enzyme and a class of particularly active catalysts bearing secondary phosphine oxide (SPO) ligands. In both cases, the key catalytic step was proposed to be intramolecular attack on a coordinated nitrile, with either an S-OH or S–O − (enzyme) or a P-OH (synthetic) nucleophile. Attack of water on the heteroatom (S or P) in the resulting metallacycle and proton transfer yields the amide and regeneratesmore »the catalyst. Evidence for this mechanism, its relevance to the formation of related metallacycles, and its potential for design of more active catalysts for nitrile hydration is summarized.« less
  4. Atomically dispersed metal catalysts have demonstrated superb electrocatalytic activity because of optimal atom efficiency. However, a rational design of low-cost, high-performance single atom catalysts remains a great challenge due to the elusive chemical reactions of the formation of metal active sites. In this work, biomass hydrogel is prepared as a template for mass production of three-dimensional carbon aerogel-supported single metal atom catalysts. By tailoring the structure of the hydrogel templates, the obtained carbon aerogels exhibit an increase of microporous defects which capture and stabilize isolated metal atoms and minimize aggregation during pyrolysis. Extended X-ray absorption fine structure, Mössbauer spectroscopy, andmore »nitrogen adsorption–desorption isotherm measurements indicate that single metal centers of FeN 4 are formed and embedded within the hierarchical porous carbon frameworks. The obtained composites exhibit outstanding catalytic activity towards oxygen reduction in alkaline media, with a high onset potential of +1.05 V and half-wave potential of +0.88 V, as well as excellent durability. Remarkably, when the carbon aerogels are used as the cathode catalyst in an aluminum–air battery, the battery achieves an ultrahigh open-circuit voltage of 1.81 V, large power density of 181.1 mW cm −2 and stable discharge voltage of 1.70 V at 20 mA cm −2 , markedly better than those with commercial Pt/C as the cathode catalyst.« less
  5. Iron single atom catalysts have emerged as one of the most active electrocatalysts towards the oxygen reduction reaction (ORR), but the unsatisfactory durability and limited activity for the oxygen evolution reaction (OER) has hampered their commercial applications in rechargeable metal–air batteries. By contrast, cobalt-based catalysts are known to afford excellent ORR stability and OER activity, due to the weak Fenton reaction and low OER Gibbs free energy. Herein, a bimetal hydrogel template is used to prepare carbon aerogels containing Fe–Co bimetal sites (NCAG/Fe–Co) as bifunctional electrocatalysts towards both ORR and OER, with enhanced activity and stability, as compared to themore »monometal counterparts. High-resolution transmission electron microscopy, elemental mapping and X-ray photoelectron spectroscopy measurements demonstrate homogeneous distributions of the metal centers within defected carbon lattices by coordination to nitrogen dopants. X-ray absorption spectroscopic measurements, in combination with other results, suggest the formation of FeN 3 and CoN 3 moieties on mutually orthogonal planes with a direct Fe–Co bonding interaction. Electrochemical measurements show that NCAG/Fe–Co delivers a small ORR/OER potential gap of only 0.64 V at the current density of 10 mA cm −2 , 60 mV lower than that (0.70 V) with commercial Pt/C and RuO 2 catalysts. When applied in a flexible Zn–air battery, the dual-metal NCAG/Fe–Co catalyst also shows a remarkable performance, with a high open-circuit voltage of 1.47 V, a maximum power density of 117 mW cm −2 , as well as good rechargeability and flexibility. Results from this study may offer an ingenious protocol in the design and engineering of highly efficient and durable bifunctional electrocatalysts based on dual metal-doped carbons.« less