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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Understanding the role of negative charge in the scaffold of an artificial enzyme for CO 2 hydrogenation on catalysis

Here, we have approached the construction of an artificial enzyme by employing a robust protein scaffold, lactococcal multidrug resistance regulator, LmrR, providing a structured secondary and outer coordination spheres around a molecular rhodium complex, [Rh I (P Et2 N gly P Et2 ) 2 ] - . Previously, we demonstrated a 2–3 fold increase in activity for one Rh-LmrR construct by introducing positive charge in the secondary coordination sphere. In this study, a series of variants was made through site-directed mutagenesis where the negative charge is located in the secondary sphere or outer coordination sphere, with additional variants made with increasingly negative charge in the outer coordination sphere while keeping a positive charge in the secondary sphere. Placing a negative charge in the secondary or outer coordination sphere demonstrates decreased activity by a factor of two compared to the wild-type Rh-LmrR. Interestingly, addition of positive charge in the secondary sphere, with the negatively charged outer coordination sphere restores activity. Vibrational and NMR spectroscopy suggest minimal changes to the electronic density at the rhodium center, regardless of inclusion of a negative or positive charge in the secondary sphere, suggesting another mechanism is impacting catalytic activity, explored in the discussion.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

A Positive Charge in the Outer Coordination Sphere of an Artificial Enzyme Increases CO 2 Hydrogenation

The protein scaffold around the active site of enzymes is known to influence catalytic activity, but specific scaffold features responsible for favorable influences are often not known. This study focuses on using an artificial metalloenzyme to probe one specific feature of the scaffold, the position of a positive charge in the outer coordination sphere around the active site. Previous work showed that a small molecular complex, [Rh(P Et 2 N glycine P Et 2 )2 ] – , immobilized covalently within a protein scaffold was activated for CO 2 hydrogenation. In this work, using an iterative design where the effect of arginine, histidine, or lysine residues placed in the outer coordination sphere of the catalytic active site were evaluated, we tested the hypothesis that positively charged groups facilitate CO 2 hydrogenation with seven unique constructs. Single-, double-, and triple-point mutations were introduced to directly compare catalytic activity, as monitored by turnover frequencies (TOFs) measured in real time with 1 H NMR spectroscopy, and evaluate related structural and electronic properties. Two of the seven constructs showed a 2- and 3-fold increase relative to the wild type, with overall rates ranging from 0.2 to 0.7 h –1 , and a crystal structure of the fastest of these shows the positive charge positioned next to the active site. A crystal structure of the arginine-containing complex shows that the arginines are positioned near the metal. Molecular dynamics (MD) studies also suggest that the positive charge is oriented next to the active site in the two constructs with faster rates but not in the others and that the positive charge near the active site holds the CO 2 near the metal, all consistent with a positive charge appropriately positioned in the scaffold benefiting catalysis. The MD studies also suggest that changes in the water distribution around the active site may contribute to catalytic activity, while modest structural changes and movement of the complex within the scaffold do not.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

A designed heme-[4Fe-4S] metalloenzyme catalyzes sulfite reduction like the native enzyme

Multielectron redox reactions often require multicofactor metalloenzymes to facilitate coupled electron and proton movement, but it is challenging to design artificial enzymes to catalyze these important reactions, owing to their structural and functional complexity. Here, we report a designed heteronuclear heme-[4Fe-4S] cofactor in cytochrome c peroxidase as a structural and functional model of the enzyme sulfite reductase. The initial model exhibits spectroscopic and ligand-binding properties of the native enzyme, and sulfite reduction activity was improved—through rational tuning of the secondary sphere interactions around the [4Fe-4S] and the substrate-binding sites—to be close to that of the native enzyme. By offering insight into the requirements for a demanding six-electron, seven-proton reaction that has so far eluded synthetic catalysts, this study provides strategies for designing highly functional multicofactor artificial enzymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Enhanced Interfacial Electron Transfer in Photocatalyst-Natural Enzyme Coupled Artificial Photosynthesis System: Tuning Strategies and Molecular Simulations

Laccase is capable of catalyzing a vast array of reactions, but its low redox potential limits its potential applications. The use of photocatalytic materials offers a solution to this problem by converting absorbed visible light into electrons to facilitate enzyme catalysis. Herein, MIL-53(Fe) and NH 2 -MIL-53(Fe) serve as both light absorbers and enzyme immobilization carriers, and laccase is employed for solar-driven chemical conversion. Electron spin resonance spectroscopy results confirm that visible light irradiation causes rapid transfer of photogenerated electrons from MOF excitation to T1 Cu(II) of laccase, significantly increasing the degradation rate constant of tetracycline (TC) from 0.0062 to 0.0127 min -1 . Conversely, there is only minimal or no electron transfer between MOF and laccase in the physical mixture state. Theoretical calculations demonstrate that the immobilization of laccase's active site and its covalent binding to the metal-organic framework surface augment the coupled system's activity, reducing the active site accessible from 27.8 to 18.1 Å. In conclusion, the constructed photo-enzyme coupled system successfully combines enzyme catalysis’ selectivity with photocatalysis's high reactivity, providing a promising solution for solar energy use.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (k cat /K M 146 M -1 s -1 ). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (k cat /K M 103,000 M -1 s -1 ) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Rescue of conformational dynamics in enzyme catalysis by directed evolution

Rational design and directed evolution have proved to be successful approaches to increase catalytic efficiencies of both natural and artificial enzymes. Protein dynamics is recognized as important, but due to the inherent flexibility of biological macromolecules it is often difficult to distinguish which conformational changes are directly related to function. Here, we use directed evolution on an impaired mutant of the proline isomerase CypA and identify two second-shell mutations that partially restore its catalytic activity. We show both kinetically, using NMR spectroscopy, and structurally, by room-temperature X-ray crystallography, how local perturbations propagate through a large allosteric network to facilitate conformational dynamics. The increased catalysis selected for in the evolutionary screen is correlated with an accelerated interconversion between the two catalytically essential conformational sub-states, which are both captured in the high-resolution X-ray ensembles. Our data provide a glimpse of an evolutionary trajectory and show how subtle changes can fine-tune enzyme function.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Unnatural biosynthesis by an engineered microorganism with heterologously expressed natural enzymes and an artificial metalloenzyme

Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here, in this work, we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium–porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Incorporation of an Asymmetric Mo−Fe−S Cluster as an Artificial Cofactor into Nitrogenase

Abstract Nitrogenase employs a sophisticated electron transfer system and a Mo−Fe−S−C cofactor, designated the M‐cluster [(cit)MoFe 7 S 9 C]), to reduce atmospheric N 2 to bioaccessible NH 3 . Previously, we have shown that the cofactor‐free form of nitrogenase can be repurposed as a protein scaffold for the incorporation of a synthetic Fe−S cluster [Fe 6 S 9 (SEt) 2 ] 4− . Here, we demonstrate the utility of an asymmetric Mo−Fe−S cluster [Cp*MoFe 5 S 9 (SH)] 3− as an alternative artificial cofactor upon incorporation into the cofactor‐free nitrogenase scaffold. The resultant semi‐artificial enzyme catalytically reduces C 2 H 2 to C 2 H 4 , and CN − into short‐chain hydrocarbons, yet it is clearly distinct in activity from its [Fe 6 S 9 (SEt) 2 ] 4− ‐reconstituted counterpart, pointing to the possibility to employ molecular design and cluster synthesis strategies to further develop semi‐artificial or artificial systems with desired catalytic activities.

Tanifuji, Kazuki↗
DOE OSTI2023

Design of supported organocatalysts from a biomass-derived difuran compound and catalytic assessment for lactose hydrolysis

The engineered structures and active sites of enzyme catalysts give rise to high catalytic activity and selectivity toward desired reactions. We have employed a biomass-derived difuran compound to append N-substituted maleimides with amino acid (glutamic acid) substitution by Diels–Alder reaction to mimic the chemical functional groups that comprise the active site channels in enzyme catalysts. The difunctionality of the biomass-derived difuran allows production of Diels–Alder adducts by appending two amino acid moieties to form a difunctional organocatalyst. The catalytic activity of the organocatalyst can be improved by immobilizing the organocatalyst on solid supporting materials. Accordingly, the structures of these immobilized organocatalysts can be engineered to mimic enzymatic active sites and to control the interaction between reactants, products, and transition states of catalytic reactions. Lactose hydrolysis was carried out to provide an example of industrial application of this approach to design and fabricate new supported organocatalysts as artificial enzymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Enzyme property prediction using artificial intelligence

Artificial intelligence (AI)-driven enzyme property prediction enables rapid discovery and engineering of enzymes for a wide range of biotechnological and therapeutic applications. Here, we first introduce the key components in AI model development, including enzyme datasets, protein representation methods, and model architectures. We then highlight a variety of AI tools developed for the prediction of enzyme properties and functional annotations, including enzyme structure, kinetic parameters, substrate specificity, thermostability, solubility, Enzyme Commission number, and Gene Ontology term. Moreover, we describe representative downstream applications enabled by these AI tools. Finally, we discuss some challenges and opportunities as well as future prospects.

Yuan, Le [University of Illinois at Urbana-Champai↗
DOE OSTI2016

Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein–staphylococcal nuclease hybrid

Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms. - Highlights: • A novel RNA restriction enzyme using SNase was developed tor cleave viral RNA. • Our enzyme cleaved influenza RNA with rates >120-fold higher rates a PIN-fusion one. • Our artificial enzyme with the L5 linker showed the highest RNA cleavage rate. • Our artificial enzyme site-selectively cleaved influenza RNA in vitro.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Artificial intelligence tools for enzyme engineering and metabolic engineering

Enzyme engineering and metabolic engineering drive innovation in energy biotechnology. In recent years, artificial intelligence (AI) has supported successful applications in designing effective enzymes and productive microbial cell factories. This review summarizes recent advances in enzyme redesign using protein language models, de novo enzyme design with generative models, and AI tools for engineering metabolism and related cellular phenotypes. Across these areas, AI models are shifting from single modality inputs to integrated representations of protein function, metabolic pathways, and cell states. We emphasize that unifying the diverse data representations across scales will be necessary for advancements in energy biotechnology.

Volk, Michael [Univ. of Illinois at Urbana-Champai↗
NASA NTRS1972

The synthesis of starch from carbon dioxide using isolubilized stabilized enzymes

Systems for artificial manufacture of starch and for delineation of technological areas, and the rationale for studying them are considered. A discussion of the enzyme-catalyzed routes of synthesis available and a choice as to the most promising route are presented. A discussion of the enzymes involved, of enzyme insolubilization technology, and of possible engineering approaches, with examples in the form of model calculations for both reactors and separators, are also presented.

Bassham, J. A.↗
DOE OSTI2012

Active Hydrogenation Catalyst with a Structured, Peptide-Based Outer-Coordination Sphere

The synthesis, catalytic activity, and structural features of a rhodium-based hydrogenation catalyst containing a phosphine ligand coupled to a 14-residue peptide are reported. Both CD and NMR spectroscopy show that the peptide adopts a helical structure in 1:1:1 TFE/MeCN/H2O that is maintained when the peptide is attached to the ligand and when the ligand is attached to the metal complex. The metal complex hydrogenates aqueous solutions of 3-butenol to 1-butanol at 360 ± 50 turnovers/Rh/h at 294 K. This peptide- based catalyst represents a starting point for developing and characterizing a peptide-based outer-coordination sphere that can be used to introduce enzyme-like features into molecular catalysts. This work was funded by the US DOE Basic Energy Sciences, Chemical Sciences, Geoscience and Biosciences Division (AJ, JCL and WJS), the Office of Science Early Career Research Program through the Office of Basic Energy Sciences (GWB, MLR and WJS). Part of the research was conducted at the W.R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by U.S. Department of Energy’s Office of Biolog-ical and Environmental Research (BER) program located at Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for the U.S. Department of Energy.

amino acid catalysts↗
DOE OSTI2016

Artificial hydrogenases based on cobaloximes and heme oxygenase

The insertion of cobaloxime catalysts in the heme-binding pocket of heme oxygenase (HO) yields artificial hydrogenases active for H 2 evolution in neutral aqueous solutions. These novel biohybrids have been purified and characterized by using UV/visible and EPR spectroscopy. These analyses revealed the presence of two distinct binding conformations, thereby providing the cobaloxime with hydrophobic and hydrophilic environments, respectively. Quantum chemical/molecular mechanical docking calculations found open and closed conformations of the binding pocket owing to mobile amino acid residues. HO-based biohybrids incorporating a {Co(dmgH) 2 } (dmgH 2 = dimethylglyoxime) catalytic center displayed up to threefold increased turnover numbers with respect to the cobaloxime alone or to analogous sperm whale myoglobin adducts. Here, this study thus provides a strong basis for further improvement of such biohybrids, using well-designed modifications of the second and outer coordination spheres, through site-directed mutagenesis of the host protein.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Experimental Definition of the S = 1 π vs S = 2 σ Reactivity and S = 2 Character in the Ground State of an S = 1 Fe IV O Complex

Iron(IV)-oxo intermediates found in iron enzymes and artificial catalysts are competent for H atom abstraction in catalytic cycles. For S = 2 intermediates, both axial and equatorial approaches are well-established. The mechanism for S = 1 sites is not as well understood: an equatorial approach is more energetically favorable, and an axial approach requires crossing from the S = 1 to the S = 2 surface. Here, in this study, we use 1s2p resonant inelastic X-ray scattering (RIXS) and Fe L-edge X-ray absorption spectroscopy on the S = 1 [Fe IV O(TMC)(CH3CN)] 2+ and observe both S = 2 and S = 1 final states, which enables the experimental evaluation of the energetics of the axial and equatorial reactivity of an S = 1 Fe IV O center on its S = 2 vs S = 1 surface. The observation of S = 2 final states in the RIXS spectrum demonstrates significant S = 2 character spin–orbit mixed into the S = 1 ground state.

energy↗