Catch-and-Release: The Assembly, Immobilization, and Recycling of Redox-Reversible Artificial Metalloenzymes
Ontology highlight
ABSTRACT:
SUBMITTER: Miller A
PROVIDER: S-EPMC10913039 | biostudies-literature | 2024 Feb
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)-phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation, and inhibitor experiments, supplemented with in-cell solid-state NMR spectroscopy, confirmed the in-cell assembly. The ArM-containing whole cells were active in the catalysis of the enantioselective Friedel-Crafts alkylation of indoles and the Diels-Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole-cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism.
| S-EPMC7986609 | biostudies-literature
Project description:Artificial metalloenzymes (ArMs) result from the incorporation of an abiotic metal cofactor within a protein scaffold. From the earliest techniques of transition metals adsorbed on silk fibers, the field of ArMs has expanded dramatically over the past 60 years to encompass a range of reaction classes and inspired approaches: Assembly of the ArMs has taken multiple forms with both covalent and supramolecular anchoring strategies, while the scaffolds have been intuitively selected and evolved, repurposed, or designed in silico. Herein, we discuss some of the most prominent recent examples of ArMs to highlight the challenges and opportunities presented by the field.
| S-EPMC6661864 | biostudies-literature
Project description:Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium-catalysed transformations, including N-H, S-H and Si-H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.
| S-EPMC5891097 | biostudies-literature
Project description:Stimuli-responsive hydrogels have a wide range of potential applications in microfluidics, which has drawn great attention. Double cross-linked hydrogels are very well suited for this application as they offer both stability and the required responsive behavior. Here, we report the integration of poly(N-isopropylacrylamide) (PNiPAAm) hydrogel with a permanent cross-linker (N,N'-methylenebisacrylamide, BIS) and a redox responsive reversible cross-linker (N,N'-bis(acryloyl)cystamine, BAC) into a microfluidic device through photopolymerization. Cleavage and re-formation of disulfide bonds introduced by BAC changed the cross-linking densities of the hydrogel dots, making them swell or shrink. Rheological measurements allowed for selecting hydrogels that withstand long-term shear forces present in microfluidic devices under continuous flow. Once implemented, the thiol-disulfide exchange allowed the hydrogel dots to successfully capture and release the protein bovine serum albumin (BSA). BSA was labeled with rhodamine B and functionalized with 2-(2-pyridyldithio)-ethylamine (PDA) to introduce disulfide bonds. The reversible capture and release of the protein reached an efficiency of 83.6% in release rate and could be repeated over 3 cycles within the microfluidic device. These results demonstrate that our redox-responsive hydrogel dots enable the dynamic capture and release of various different functionalized (macro)molecules (e.g., proteins and drugs) and have a great potential to be integrated into a lab-on-a-chip device for detection and/or delivery.
| S-EPMC8780672 | biostudies-literature
Project description:Three-helix bundles and coiled-coil motifs are well-established de novo designed scaffolds that have been investigated for their metal-binding and catalytic properties. Satisfaction of the primary coordination sphere for a given metal is sufficient to introduce catalytic activity and a given structure may catalyze different reactions dependent on the identity of the incorporated metal. Here we describe recent contributions in the de novo design of metalloenzymes based on three-helix bundles and coiled-coil motifs, focusing on non-heme systems for hydrolytic and redox chemistry.
| S-EPMC4380837 | biostudies-literature
Project description:The biotechnological revolution has made it possible to create enzymes for many reactions by directed evolution. However, because of the immense number of possibilities, the availability of enzymes that possess a basal level of the desired catalytic activity is a prerequisite for success. For new-to-nature reactions, artificial metalloenzymes (ARMs), which are rationally designed hybrids of proteins and catalytically active transition-metal complexes, can be such a starting point. This Account details our efforts toward the creation of ARMs for the catalysis of new-to-nature reactions. Key to our approach is the notion that the binding of substrates, that is, effective molarity, is a key component to achieving large accelerations in catalysis. For this reason, our designs are based on the multidrug resistance regulator LmrR, a dimeric transcription factor with a large, hydrophobic binding pocket at its dimer interface. In this pocket, there are two tryptophan moieties, which are important for promiscuous binding of planar hydrophobic conjugated compounds by π-stacking. The catalytic machinery is introduced either by the covalent linkage of a catalytically active metal complex or via the ligand or supramolecular assembly, taking advantage of the two central tryptophan moieties for noncovalent binding of transition-metal complexes. Designs based on the chemical modification of LmrR were successful in catalysis, but this approach proved too laborious to be practical. Therefore, expanded genetic code methodologies were used to introduce metal binding unnatural amino acids during LmrR biosynthesis in vivo. These ARMs have been successfully applied in Cu(II) catalyzed Friedel-Crafts alkylation of indoles. The extension to MDRs from the TetR family resulted in ARMs capable of providing the opposite enantiomer of the Friedel-Crafts product. We have employed a computationally assisted redesign of these ARMs to create a more active and selective artificial hydratase, introducing a glutamate as a general base at a judicious position so it can activate and direct the incoming water nucleophile. A supramolecularly assembled ARM from LmrR and copper(II)-phenanthroline was successful in Friedel-Crafts alkylation reactions, giving rise to up to 94% ee. Also, hemin was bound, resulting in an artificial heme enzyme for enantioselective cyclopropanation reactions. The importance of structural dynamics of LmrR was suggested by computational studies, which showed that the pore can open up to allow access of substrates to the catalytic iron center, which, according to the crystal structure, is deeply buried inside the protein. Finally, the assembly approaches were combined to introduce both a catalytic and a regulatory domain, resulting in an ARM that was specifically activated in the presence of Fe(II) salts but not Zn(II) salts. Our work demonstrates that LmrR is a privileged scaffold for ARM design: It allows for multiple assembly methods and even combinations of these, it can be applied in a variety of different catalytic reactions, and it shows significant structural dynamics that contribute to achieving the desired catalytic activity. Moreover, both the creation via expanded genetic code methods as well as the supramolecular assembly make LmrR-based ARMs highly suitable for achieving the ultimate goal of the integration of ARMs in biosynthetic pathways in vivo to create a hybrid metabolism.
| S-EPMC6427492 | biostudies-literature
Project description:Peptide and low molecular weight amino acid-based materials that self-assemble in response to environmental triggers are highly desirable candidates in forming functional materials with tunable biophysical properties. In this paper, we explore redox-sensitive self-assembly of cationic phenylalanine derivatives conjugated to naphthalene diimide (NDI). Self-assembly of the cationic Phe-NDI conjugates into nanofibrils was induced in aqueous solvent at high ionic strength. Under reducing conditions, these self-assembled Phe-NDI conjugate fibrils underwent a morphological change to non-fibril aggregates. Upon reoxidation, the initially observed fibrils were reformed. The study herein provides an interesting strategy to effect reversible switching of the structure of supramolecular materials that can be applied to the development of sophisticated stimulus-responsive materials.
| S-EPMC5665789 | biostudies-literature
Project description:Artificial metalloenzymes (ArMs), created by introducing synthetic cofactors into protein scaffolds, are an emerging class of catalyst for non-natural reactions. Although many classes of ArMs are known, in vitro reconstitution of cofactors and proteins has been a limiting step in the high-throughput screening and directed evolution of ArMs because purification of individual host proteins is time-consuming. We describe the application of a platform to combine mutants of the P450 enzyme CYP119 and the cofactor Ir(Me)MPIX in vivo, by coexpression of the CYP119 mutants with the heme transporter encoded by the hug operon, to the directed evolution of ArMs containing Ir(Me)MPIX in whole cells. We applied this platform to the development an ArMs catalyzing the insertion of the acyclic carbene from α-diazopropanoate esters (Me-EDA) into the N-H bonds of N-alkyl anilines, a combination of carbene and amine classes for which mutant enzymes of natural hemoproteins previously reacted with low enantioselectivity. The mutants of the artificial metalloenzyme Ir(Me)CYP119 identified by an evolution campaign involving more than 4000 mutants are shown to catalyze the reaction of Me-EDA with N-methyl anilines to form chiral chiral amino esters with high TON and good enantioselectivity, thereby demonstrating that the directed evolution of ArMs can rival that of natural enzymes in vivo.
| S-EPMC9707807 | biostudies-literature
Project description:Many efforts are being made in the design and engineering of metalloenzymes with catalytic properties fulfilling the needs of practical applications. Progress in this field has recently been accelerated by advances in computational, molecular and structural biology. This review article focuses on the recent examples of oxygen-activating metalloenzymes, developed through the strategies of de novo design, miniaturization processes and protein redesign. Considerable progress in these diverse design approaches has produced many metal-containing biocatalysts able to adopt the functions of native enzymes or even novel functions beyond those found in Nature.
| S-EPMC5021598 | biostudies-literature
Project description:The reductive coupling of aldehydes and alkenes is an emerging technology that holds the potential to reinvent carbonyl addition chemistry. However, existing enantioselective methods are limited to form "branched" products. Herein, we present a directed enantio- and diastereoselective alkylation of aldehydes with simple olefins to selectively yield linear coupling products. This is achieved by redox-neutral remote functionalization, whereby a tethering "catch-release" strategy decisively solves the key problems of reactivity and selectivity.
| S-EPMC6342409 | biostudies-literature