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14 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Circularity in Sequence-Controlled Copolyamides Enabled by Regioselective Enzymatic Hydrolysis

Sequence-controlled polymers enable precise control over macromolecular structures and function, but both their synthesis and end-of-life management remain fundamental challenges. Achieving high sequence fidelity is synthetically demanding, and conventional depolymerization methods lack regioselectivity, leading to irreversible loss of encoded molecular information and limiting polymer circularity. Enzymatic catalysis offers a potential solution by combining substrate specificity with selective bond cleavage. Here, we report the synthesis, characterization, and regioselective enzymatic depolymerization of poly- (X,AMA), a sequence-controlled copolyamide composed of alternating hexamethylenediamine−adipic acid (MA) and pxylylenediamine− adipic acid (XA) repeat units. Poly(X,AMA) was synthesized via solid-state polycondensation (SSP) of sequence-defined oligomers, enabling precise control over repeat-unit order. Polymer microstructure and sequence fidelity were confirmed by 13 C NMR spectroscopy and MALDI−TOF mass spectrometry. Comparison with a statistical copolymer analogue and Nylon-66 demonstrated pronounced differences in crystallinity, morphology, and thermal behavior arising from sequence control. Screening of 96 Nylon hydrolase homologues against poly(X,AMA) revealed strongly enzyme-dependent depolymerization profiles. While tetrad formation was generally favored, enzymes displayed pronounced sequence selectivity, preferentially releasing distinct sequence-defined tetrads XAMA or MAXA. SSP of sequence-defined tetrad MAXA produced a copolyamide with near identical monomer ordering as poly(X,AMA). Computational modeling of enzyme−substrate complexes identified structural features consistent with the observed regioselectivity. Together, these results establish selective enzymatic depolymerization as a viable strategy for the circular recycling of sequence-controlled polymers and provide a foundation for the rational engineering of enzymes for programmable polymer deconstruction.

Amides↗
DOE OSTI2026

Discovery and engineering of enzymes for new-to-nature photobiocatalysis

Photobiocatalysis integrates enzymatic catalysis with photochemistry, enabling challenging radical transformations with high selectivity under mild conditions. Early developments in this field were largely driven by the discovery that enzyme-bound cofactors can form photoactive charge–transfer complexes with substrates, thereby initiating radical chemistry upon light irradiation. Recent advances, however, have substantially expanded the mechanistic landscape of photobiocatalysis through diverse mechanisms. This review summarizes major developments in photobiocatalysis reported since 2024. Rather than cataloging individual reactions, we focus on the fundamental mechanisms of radical generation and interception within enzyme active sites, and discuss how these mechanistic principles guide the discovery, engineering, and design of enzymes for new-to-nature photobiocatalysis.

Bai, Zibo [University of Illinois Urbana-Champaign↗
DOE OSTI2025

H-cluster Intermediates and Catalytic Properties of Clostridium pasteurianum [FeFe]-Hydrogenase III

[FeFe]-Hydrogenases are structurally diverse enzymes that catalyze reversible H2 activation at a catalytic cofactor or H-cluster. The H-cluster is a [4Fe-4S] cubane linked by a cysteine thiolate to a diiron subsite containing unique CO, CN-, and dithiomethylamine ligands. The established H-cluster resting state of [4Fe-4S]2+-[FeII-FeI], or Hox, functions in H2 binding and oxidation, or by proton-coupled reduction initiates H2 evolution. In contrast, in Clostridium pasteurianum [FeFe]-hydrogenase III (CpIII) the resting state of the H-cluster is fully oxidized, [4Fe-4S]2+-[FeII-FeII], or Hox+1. To begin to understand if Hox+1 has a role in the mechanism of CpIII, we determined the spectroscopic and redox properties of CpIII H-cluster states under catalytic conditions. CpIII poised in Hox+1 and either equilibrated under 1 atm of H2 or reduced with sodium dithionite, resulted in a mixture of reduced states including Hox (Em8 = -407 mV), Htrans-like [4Fe-4S]+-[FeII-FeII] (Em8 = -418 mV), Hred [4Fe-4S]+-[FeII-FeI], and HredH+ [4Fe-4S]2+-[FeI-FeI] (Em8 = -455-480 mV). Under H2 the population of the Htrans-like state was >20-fold higher than Hox, implicating a role in CpIII catalysis. Unlike other enzymes, there was no spectral evidence of fully reduced states, such as HsredH+ ([4Fe-4S]+-[FeI-FeI]) or Hhyd ([4Fe-4S]+-[FeII-FeII]-H-). Thus, while the H-cluster states of CpIII encompass most of the catalytic intermediates, it is either unable to form HsredH+ and Hhyd, or these states are highly destabilized in CpIII. Thus, these results demonstrate that catalytic intermediates of reduced CpIII differ from the typical intermediates of other catalytic [FeFe]-hydrogenases and may explain the catalytic preference for H2 production.

08 HYDROGEN↗
DOE OSTI2025

Conformational ensembles reveal the origins of serine protease catalysis

Enzymes exist in ensembles of states that encode the energetics underlying their catalysis. Conformational ensembles built from 1231 structures of 17 serine proteases revealed atomic-level changes across their reaction states. By comparing the enzymatic and solution reaction, we identified molecular features that provide catalysis and quantified their energetic contributions to catalysis. Serine proteases precisely position their reactants in destabilized conformers, creating a downhill energetic gradient that selectively favors the motions required for reaction while limiting off-pathway conformational states. The same catalytic features have repeatedly evolved in proteases and additional enzymes across multiple distinct structural folds. In conclusion, our ensemble-function analyses revealed previously unknown catalytic features, provided quantitative models based on simple physical and chemical principles, and identified motifs recurrent in nature that may inspire enzyme design.

Du, Siyuan [Stanford Univ., CA (United States)] (O↗
DOE OSTI2025

The role of Tyr34 in proton coupled electron transfer and product inhibition of manganese superoxide dismutase

Human manganese superoxide dismutase (MnSOD) plays a crucial role in controlling levels of reactive oxygen species (ROS) by converting superoxide (O$^{•–}_{2}$) to molecular oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ) with proton-coupled electron transfers (PCETs). A key catalytic residue, Tyr34, determines the activity of human MnSOD and also becomes post-translationally inactivated by nitration in various diseases associated with mitochondrial dysfunction. Tyr34 has an unusual pK a due to its proximity to the Mn metal and undergoes cyclic deprotonation and protonation events to promote the electron transfers of MnSOD. Neutron diffraction, X-ray spectroscopy, and quantum chemistry calculations in oxidized, reduced and product inhibited enzymatic states shed light on the role of Tyr34 in MnSOD catalysis. The data identify the contributions of Tyr34 in MnSOD activity that support mitochondrial function and give a thorough characterization of how a single tyrosine modulates PCET catalysis. Product inhibition occurs by an associative displacement mechanism.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Polymer Deconstruction and Redesign Strategies for Plastics Recycling

Advancing plastics recycling requires both the selective deconstruction of existing polymers and the design of new materials that enable efficient reuse without loss of performance. This perspective highlights an integrated approach that is rooted in polymer chemistry, catalysis, and process engineering which can enable a circular plastics economy. Here, we outline recent advances in catalytic, solvolytic, and enzymatic pathways for plastic deconstruction, and examine the molecular design principles driving next-generation recyclable-by-design and bio-based polymers. Despite these advances, major knowledge gaps remain in understanding the evolution of polymer morphology and catalyst structure during deconstruction, assessing deconstruction processes with realistic polymers, and offering redesigned polymers with competitive cost and environmental advantage over conventional plastics. United States Department of Energy (U.S. DOE) national laboratories offer unique capabilities to address these challenges through in situ and operando characterization, high-throughput experimentation, environmental studies, technoeconomic and life cycle assessment, scale-up support, and collaboration networks. Advances made in understanding plastic deconstruction mechanisms and structure-property correlations of redesigned polymers inform emerging research directions including autonomous experimentation, real-time feedback-enabled process optimization, and protein engineering for enzymatic depolymerization.

36 MATERIALS SCIENCE↗
DOE OSTI2026

Data for Photoenzymatic Stereoablative Enantioconvergence of γ-chiral Oximes via Hydrogen Atom Transfer

Producing enantioenriched molecules from racemic mixtures is essential for manufacturing. Traditional methods such as resolution, deracemization and enantioconvergent catalysis primarily involve separating or converting enantiomers without altering their structures, or functionalization of stereocentres at or proximal to functional groups. However, there are challenges in enantioselectively forging C–H bonds that are remote from functional groups via hydrogen atom transfer (HAT) with these methods. Here we introduce a strategy for the photoenzymatic stereoablative enantioconvergence of γ-chiral oximes using repurposed flavin-dependent ene-reductases. A photoinduced single-electron reduction of the γ-chiral oxime by an ene-reductase generates an iminyl radical, which then undergoes stereoablative 1,5-HAT at the γ-stereocentre. Subsequent chiral reconstruction through enzymatic HAT and spontaneous imine hydrolysis yields the γ-chiral ketone with high enantioselectivity. This work provides a robust method for remote stereoablative enantioconvergent HAT and broadens the synthetic utility of photobiocatalysis.

Bioproducts↗
DOE OSTI2025

Insights from designing an artificial cascade catalysis system using principles from substrate channeling in enzymes

Generalizing the key requirements of highly-selective, multi-step chemical conversions involving spatially separated reaction centers remains one of the grand challenges of chemistry. Much work towards this effort has focused on decomposing multi-step conversions into their constituent reactions, whose intermediates are successively upgraded in a chemical cascade via diffusion from center to center. This approach for synthesizing more complex molecules takes its cues from biochemical networks, where near-unit conversion of even complex carbohydrates is achieved by upgrading chemical precursors via enzymatic cascades. In this computational study we examine a simple cascade involving coupled Ag and Cu catalysts that sequentially converts CO2 to CO and then CO2 and CO to reduced products, generically named CO2Product and COProduct. The system architecture is inspired by the phenomenon of biological substrate channeling, and components are examined to evaluate their effects on conversion efficiency in the cascade. Aside from a substrate channel linking two reaction centers, we find efficient cascades must also incorporate directional substrate diffusion, compartmentalization of the reaction centers, and proper timing of substrate arrival at the active center. We make explicit linkages between these requirements and chemical conversion in known biological systems, revealing additional control elements that could be incorporated.

CO 2 reduction↗
DOE OSTI2026

Determinants of Stereoselectivity in Monoterpene Synthases

Monoterpene synthases (MTSs) catalyze the conversion of an achiral prenyl diphosphate precursor, most commonly geranyl diphosphate (GPP), into structurally and stereochemically diverse products. However, remains poorly understood. Here, we combine enzymatic assays with six MTSs and selected variants, along with extensive molecular dynamics simulations to the mechanistic basis of stereoselectivity. We demonstrate that the preferred helical binding conformation of GPP, determined from free-energy calculations and selected crystal structures of MTSs, correlates well with the experimentally determined stereoselectivity of MTSs, whereas only a poor correlation is observed between the binding of enantiomers of linalyl diphosphate (LPP), a chiral intermediate of MTS catalysis, and the stereochemical reaction outcomes. Free energy maps indicate that enzyme-bound GPP conformers preferentially occupy regions that enable a direct and stereochemically faithful transition from GPP to subsequent carbocations. In contrast, LPP frequently populates regions of the energy surface where conformational scrambling can occur, thus leading to a lack of correlation between LPP enantiomer binding and product stereochemistry. Overall, these findings establish that the configuration of GPP is the primary determining factor for stereochemical outcomes in MTSs, offering a new framework for designing stereoselective terpene synthases.

Srividya, Narayanan (ORCID:0000000179347987)↗
DOE OSTI2025

Structural basis of undecaprenyl phosphate glycosylation leading to polymyxin resistance in Gram-negative bacteria

In Gram-negative bacteria, the enzymatic modification of Lipid A with aminoarabinose (L-Ara4N) leads to resistance against polymyxin antibiotics and cationic antimicrobial peptides. ArnC, an integral membrane glycosyltransferase, attaches a formylated form of aminoarabinose to the lipid undecaprenyl phosphate, enabling its association with the bacterial inner membrane. Here, we present cryo-electron microscopy structures of ArnC from S. enterica in apo and nucleotide-bound conformations. These structures reveal a conformational transition that takes place upon binding of the partial donor substrate. Using coarse-grained and atomistic simulations, we provide insights into substrate coordination before and during catalysis, and we propose a catalytic mechanism that may operate on all similar metal-dependent polyprenyl phosphate glycosyltransferases. The reported structures provide a new target for drug design aiming to combat polymyxin resistance.

Ashraf, Khuram U. [Rutgers University, Newark, NJ ↗
DOE OSTI2026

"Lignin First" Catalytic Biomass Fractionation: Cooperative Research and Development (Final Report)

In this work, a continuous zwitterionic chromatography using simulated moving beds will be conducted to separate Li and Mg from brines. The existing program that ExxonMobil is carrying out with Clariant and Genomatica is consistent with all pioneer cellulosic fuels plants inasmuch as biomass enters the facility and is processed, in its entirety, through at least the hydrolysis step of biochemical conversion. This approach creates significant operating challenges as biomass can vary widely in composition and mechanical properties across and within a given biomass type. The proposed program involves a first step fractionation using solvolysis and reductive catalysis to generate a lignin oil containing well-defined monomers, dimers, and oligomers and a delignified cellulose/ hemicellulose pulp. The amount of lignin oil vs pulp and the type of monomers produced are expected to vary with biomass type due to differences in lignin composition and chemistry.

09 BIOMASS FUELS↗
DOE OSTI2026

Regulation of Energy and Mass Transport in a Hydrogen-Bonded Framework for Visible-Light-Driven CO2 Reduction in Water

Photoenzymatic reduction of CO2 to formate is a promising strategy for carbon valorization, yet its efficiency is still limited by inefficient energy and mass transport. Here, we design a series of isostructural hydrogen-bonded organic frameworks (HOFs) that establish confinement effects to promote photocatalytic NADH regeneration and the subsequent NADH-dependent enzymatic CO2-to-formate reduction. We demonstrate that spatial confinement within the framework channels localizes exciton migration to nanoscale domains and promotes interfacial dissociation. Additionally, Rh-induced electronic-structure modulation enables ultrafast electron transfer, while the intrinsic hydrogen-bond network furnishes directional proton conduction to NAD+. These synergistic regulations afford a photocatalytic NADH regeneration efficiency of 99.8% with a record apparent quantum efficiency of 32.8%, and drive formate production at a rate of 3020 μmol g-1 h-1 with 100% selectivity─the highest rate reported to date for all light-driven systems in water. The HOF-based catalyst retains 86.3% of its initial activity over five cycles, highlighting its robustness. This work offers mechanistic insight into how microenvironment engineering within HOF architectures regulates energy and mass transport in photoenzymatic catalysis, paving the way for the rational design of advanced hybrid catalytic systems.

Xu, Jiaxing↗
DOE OSTI2026

Structural insights into RNase H catalytic mechanism from room-temperature X-ray and neutron crystallography of apo- and RNA/DNA hybrid-bound enzyme

RNase H enzymes are sequence-nonspecific endonucleases that cleave RNA strands in RNA/DNA hybrid duplexes, an enzymatic process essential in DNA replication and repair in both prokaryotes and eukaryotes. Also, RNase H activity of the reverse transcriptase in human immunodeficiency viruses (HIV-1 and HIV-2) is indispensable for the viral replication cycle. RNase H enzymes play an central role in the development of gene therapies and are targets for novel antivirals. It is therefore of great importance to gain a detailed understanding of the RNase H catalytic mechanism to improve drug design. We utilized Bacillus halodurans RNase H1 (BhRNase H1) to shed light on its function and catalytic mechanism. Room-temperature neutron crystallography of the wild-type and inactive D132N mutant enzymes revealed that E109, belonging to the catalytic DEDD motif, can change its protonation state, allowing us to propose its role in the protonation of the leaving O3′ hydroxyl group of RNA. X-ray crystallography has demonstrated the ability of the RNA/DNA duplex to slide along the protein surface upon metal ion binding at site M A , transforming a product mimic into a Michaelis-like complex, which confirms an essential role of the M A metal ion in catalysis.

Enzyme mechanisms↗
DOE OSTI2025

Chaotrope-Based Approach for Rapid In Vitro Assembly and Loading of Bacterial Microcompartment Shells

Bacterial microcompartments (BMCs) are proteinaceous organelles that self-assemble into selectively permeable shells that encapsulate enzymatic cargo. BMCs enhance catalytic pathways by reducing crosstalk among metabolites, preventing harmful intermediates from leaking into the cytosol and increasing reaction efficiency via enzyme colocalization. The intrinsic properties of BMCs make them attractive for biotechnological engineering. However, in vivo expression methods for shell synthesis have significant drawbacks that limit the potential design space for these nanocompartments. Here, we describe the development of an efficient and rapid method for the in vitro assembly of BMC shells from their protein building blocks. Our method enables large-scale construction of BMC shells by utilizing urea as a chaotropic agent to control self-assembly and provides an approach for encapsulation of both biotic and abiotic cargo under a broad range of reaction conditions. We demonstrate an enhanced level of control over the assembly of BMC shells in vitro and expand the design parameter space for engineering BMC systems with specialized and enhanced catalytic properties.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗