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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 307 records · Page 17
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

The N-terminus of varicella-zoster virus glycoprotein B has a functional role in fusion

Varicella-zoster virus (VZV) is a medically important alphaherpesvirus that induces fusion of the virion envelope and the cell membrane during entry, and between cells to form polykaryocytes within infected tissues during pathogenesis. All members of the Herpesviridae , including VZV, have a conserved core fusion complex composed of glycoproteins, gB, gH and gL. The ectodomain of the primary fusogen, gB, has five domains, DI-V, of which DI contains the fusion loops needed for fusion function. We recently demonstrated that DIV is critical for fusion initiation, which was revealed by a 2.8Å structure of a VZV neutralizing mAb, 93k, bound to gB and mutagenesis of the gB-93k interface. To further assess the mechanism of mAb 93k neutralization, the binding site of a non-neutralizing mAb to gB, SG2, was compared to mAb 93k using single particle cryogenic electron microscopy (cryo-EM). The gB-SG2 interface partially overlapped with that of gB-93k but, unlike mAb 93k, mAb SG2 did not interact with the gB N-terminus, suggesting a potential role for the gB N-terminus in membrane fusion. The gB ectodomain structure in the absence of antibody was defined at near atomic resolution by single particle cryo-EM (3.9Å) of native, full-length gB purified from infected cells and by X-ray crystallography (2.4Å) of the transiently expressed ectodomain. Both structures revealed that the VZV gB N-terminus (aa72-114) was flexible based on the absence of visible structures in the cryo-EM or X-ray crystallography data but the presence of gB N-terminal peptides were confirmed by mass spectrometry. Notably, N-terminal residues 109 KSQD 112 were predicted to form a small α-helix and alanine substitution of these residues abolished cell-cell fusion in a virus-free assay. Importantly, transferring the 109 AAAA 112 mutation into the VZV genome significantly impaired viral propagation. These data establish a functional role for the gB N-terminus in membrane fusion broadly relevant to the Herpesviridae .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Developing Advanced Genetic and Synthetic Biology Tools for Improved Algae Productivity

Under the UCSD-led Productivity Enhanced Algae and ToolKits (PEAK) project we sought to develop tools to enable the production of valuable co-products, as well as strategies to decrease the cost of cultivation, with an intended outcome of enabling the economic production of algae-based biofuel. We have developed new genetic tools and high throughput selection methods for eukaryotic green algae and used them to express a high value recombinant protein; a growth factor known as Osteopontin (OPN). Recombinant OPN protein expression levels were improved through a newly developed rapid breeding and mutagenesis strain improvement method to generate a robust strain capable of growing in outdoors raceway conditions in brackish water that tolerated high pH and large temperature swings. We further applied the new genetic tools to express recombinant protein in a recently bioprospected strain with even more robust growth characteristics. In response to the COVID-19 pandemic we used our improved workflow to rapidly express, purify, and characterize a recombinant SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and demonstrate that it functionally interacts with its cognate human host receptor ACE2. The newly identified extremophile strain Chlamydomonas sp (402), both mating type (mt+) and mating type (mt-), have been deposited in the Chlamydomonas Resource Center (https://www.chlamycollection.org/), making them available world-wide with no restrictions. In addition, all of genetic tools are also deposited at this site and are also available world-wide with no restrictions.

09 BIOMASS FUELS↗
DOE OSTI2023

Improved Biofuel Production through Discovery and Engineering of Terpene Metabolism in Switchgrass

Project Objectives - Of the myriad specialized metabolites that plants deploy to adapt to environmental challenges, terpenes form the largest group. In many major crops, unique terpene blends serve as key stress defenses that directly impact plant fitness and yield. In addition, terpenes, such as bisabolene and pinene, are used for producing renewable biofuels. Essential to advancing a broader use of terpenes for biofuel feedstock engineering is a system-wide knowledge of the diverse biosynthetic machinery and defensive potential of often species-specific terpene blends. The proposed project would merge genome-wide enzyme discovery with comparative –omics, protein structural and plant microbiome studies to define the biosynthesis and stress-defensive functions of the switchgrass (Panicum virgatum) terpene network. These insights would be combined with developing and applying non-transgenic genome editing tools to design plants with desirable terpene blends for higher productivity and biofuel production on marginal lands. As a dedicated lignocellulosic feedstock for U.S. biofuel production with high net energy yield, stress tolerance, and available genome resources, switchgrass is well-suited for devising new avenues for biofuel production. Project Description – The diversity of plant terpene defenses is governed by species-specific families of terpene synthase (TPS) and cytochrome P450 monooxygenase (P450) enzymes. Mining of the switchgrass genome (genotype Alamo) identified ~100 TPS and P450 candidate genes, and combinatorial biochemical analysis of synthesized TPSs and P450s revealed more than a dozen enzymes with common and novel activities. In addition, several identified terpene metabolites and the corresponding transcripts were up-regulated in response to abiotic stressors. These findings demonstrate a unique switchgrass terpene network with probable importance to abiotic stress tolerance, thus providing a large chemical portfolio for optimizing crop resistance, yield, and biofuel composition. Leveraging these preliminary data, we propose to generate a genome-wide map of the switchgrass terpene metabolic network through multi-gene co-expression analyses that allow the efficient cross-validation of TPS and P450 functions. Key enzymes would further be applied to structure-function studies via X-ray protein crystallography, homology modeling and site-directed mutagenesis to gain mechanistic insight into the catalytic specificity of switchgrass terpene metabolism and provide gene and amino acid targets for genome editing. In tandem with terpene pathway discovery, system-wide metabolomics, transcriptomics and proteomics studies in switchgrass accessions of contrasting drought tolerance would define the role of switchgrass terpene metabolism in conferring abiotic stress resilience. Metabolic changes would be assessed in a combined approach of targeted (terpenes) and untargeted metabolite profiling using a high-resolution LC-MS/MS approach, differential gene expression analyses through multiplexed Illumina RNA sequencing, and quantitative analysis of high-priority pathway enzymes using multiple reaction monitoring (MRM). Drawing on these insights, knock-down/out mutants of stress-associated pathway nodes would be generated by optimizing transient virus-induced gene silencing (VIGS) and CRISPR/Cas9 systems under control of the Tobacco Rattle Virus (TRV). The resulting mutant lines would then be analyzed for stress susceptibility and the impact on the root microbiome to define gene functions in planta. Knowledge of terpene pathways, enzyme mechanisms and bioactivities would be applied to enhance switchgrass stress resilience and to tailor-make terpene blends for biofuel production. Here, TRV-enabled CRISPR/Cas9 genome editing, including allele-specific knock-out of redundant genes, engineering of enzyme specificity via structure-guided point mutations, and overexpression of terpene genes relevant to stress-protection or biofuel production, would be used to increase metabolic flux toward desired pathways. Broader Impacts - Integrating the system-wide discovery, mechanistic analysis and non-transgenic genome engineering of the switchgrass terpene network aligns the required steps to unlock the chemical potential of this important metabolite class to generate crops that are more resistant to stress and provide advanced biofuel production in light of rising climate pressures as foreseeable challenges for bioenergy crop cultivation. The proposed project would further offer interdisciplinary student training through active involvement in the project and integration of research concepts and outcomes into newly-developed graduate and undergraduate courses on Plant Biotechnology.

09 BIOMASS FUELS↗
DOE OSTI2024

Mechanistic Studies of a Primitive Homolog of Nitrogenase Involved in Coenzyme F430 Biosynthesis

Methyl-coenzyme M reductase (MCR) is the key enzyme in the biological formation and anaerobic oxidation of methane (AOM). Methane is a potent greenhouse gas and the major component of natural gas. Given the abundance of natural gas reserves in remote areas, there is great current interest in a scalable bio-based process for the conversion of methane to liquid fuel or other high-value commodity chemicals. MCR holds much promise for use in such a methane bioconversion strategy. However, MCR cannot currently be produced in an active form in a heterologous host, due in large part to the lack of genetic and biochemical information about the production of holo MCR. In an effort to overcome this deficiency, our laboratory elucidated the biosynthetic pathway of the unique nickel-containing coenzyme of MCR, F430. The key step in coenzyme F430 biosynthesis (Cfb) was found to involve an unprecedented reductive cyclization reaction that converts Ni-sirohydrochlorin a , c -diamide to 15,17 3 -seco-F430-17 3 -acid. This remarkable transformation, which involves a 6-electron reduction of the isobacteriochlorin ring system, cyclization of the c -acetamide side chain to form a γ-lactam ring, and the formation of 7 stereocenters, is catalyzed by a primitive homolog of nitrogenase (CfbCD). Nitrogenase is a two-component metalloenzyme that catalyzes the ATP-dependent reduction of dinitrogen to ammonia (nitrogen fixation). Homologs of nitrogenase are also involved in the biosynthesis of the photosynthetic pigments chlorophyll and bacteriochlorophyll. Phylogenetic analysis of the CfbCD complex suggests that it is representative of a more ancient lineage of the nitrogenase superfamily, and a thorough investigation of its structure and function is likely to shed light on the mechanisms and evolution of these important metalloenzymes that catalyze multi-electron redox reactions. Moreover, a detailed understanding of the mechanism of the CfbCD complex may aid in the development of specific inhibitors to help reduce natural greenhouse gas emissions and can be exploited for the heterologous production of MCR for methane bioconversion. Towards these goals, the following Specific Aims will be pursued to determine the: 1) Identity of the CfbCD reaction product. The exact reaction catalyzed by CfbCD, including the number of electrons transferred and whether it involves enzymatic cyclization, is unclear. Several approaches, including reaction stoichiometry measurements, spectroelectrochemistry, and magnetic resonance spectroscopy will be applied to elucidate the structure of the reaction product and establish whether CfbCD is a reductase or reductive cyclase. 2) Structure, conformational dynamics, and oligomerization state changes of CfbCD. Significant insight into the mechanism and allosteric regulation of CfbCD can be obtained by assessing changes in the structure and dynamics of the complex during the catalytic cycle. To accomplish this, a combination of size-exclusion chromatography, hydrogen-deuterium exchange mass spectrometry, molecular dynamics simulations, and high-resolution structural methods will be employed. 3) Source, order, and stereochemistry of proton additions during CfbCD catalysis. Details regarding the order and stereochemistry of proton additions during the CfbCD reaction will be uncovered using a combined spectroscopic and computational approach. Complementary mechanistic studies employing site-directed mutagenesis and substrate analogs will establish the identity of active site acid residues and the possible involvement of substrate-assisted catalysis during the CfbCD reaction.

09 BIOMASS FUELS↗
DOE OSTI2026

Rational Design of Lanmodulin Variants for Size-Based Selectivity of Individual Rare Earth Elements

Rare earth elements (REEs) are essential to modern technologies, yet their high physical and chemical similarity makes separation of individual REEs difficult and environmentally taxing. Metalloproteins offer a promising alternative for selective REE binding, as they tend to have high metal ion affinity and specificity. Lanmodulin (LanM), in particular, has arisen as a potential candidate for REE separation as it exhibits picomolar affinity for elements in the REE family. Prior work has shown that the single point mutation D9N can shift LanM’s preference away from lanthanides toward actinides, motivating efforts to tune selectivity of LanM through targeted mutagenesis. Here, we tested the hypothesis that introducing selective aspartic acid to glutamic acid substitutions in the metal coordinating EF hands of LanM would impose steric constraints that would drive LanM affinity away from larger ions, such as La3+, to smaller ions, such as Y3+. To test this hypothesis, a combination of computational and experimental approaches were employed to evaluate the signal mutations LanM D5E and LanM D3E and the double mutants LanM D1ED5E and LanM D3ED9E. Surprisingly, increasing the number of mutations within the metal center did not enhance affinity for smaller REEs, or decrease affinity for larger ions. Only the single point mutation LanM D5E weakened La3+ binding by one order of magnitude relative to LanM wild type (WT), and pairing it with a second mutation to produce LanM D1ED5E drove La3+ affinity to be stronger than that seen for LanM WT. The D3E mutation alone prevented proper expression and folding, but paring it with D9E to produce LanM D3ED9E rescued expression and yielded La3+ affinities comparable to LanM WT. All variants that expressed (LanM D5E, LanM D1ED5E, LanM D3ED9E) displayed Y3+ affinities comparable to LanM WT. Overall, these results highlight the tunability of LanM’s metal-binding environment but also expose current limitations in predicting structural responses to point mutations within a protein sequence. This work establishes a foundation that can be used for refining computational and experimental strategies to engineer metalloproteins with tailored REE selectivity.

Close, Emily [Pacific Northwest National Laborator↗
DOE OSTI2024

Harnessing genetic engineering to drive economic bioproduct production in algae

Our reliance on agriculture for sustenance, healthcare, and resources has been essential since the dawn of civilization. However, traditional agricultural practices are no longer adequate to meet the demands of a burgeoning population amidst climate-driven agricultural challenges. Microalgae emerge as a beacon of hope, offering a sustainable and renewable source of food, animal feed, and energy. Their rapid growth rates, adaptability to non-arable land and non-potable water, and diverse bioproduct range, encompassing biofuels and nutraceuticals, position them as a cornerstone of future resource management. Furthermore, microalgae’s ability to capture carbon aligns with environmental conservation goals. While microalgae offers significant benefits, obstacles in cost-effective biomass production persist, which curtails broader application. This review examines microalgae compared to other host platforms, highlighting current innovative approaches aimed at overcoming existing barriers. These approaches include a range of techniques, from gene editing, synthetic promoters, and mutagenesis to selective breeding and metabolic engineering through transcription factors.

09 BIOMASS FUELS↗
DOE OSTI2022

From Farm to Flight: CoverCress as a Low Carbon Intensity Cash Cover Crop for Sustainable Aviation Fuel Production. A Review of Progress Towards Commercialization

Thlaspi arvense L. (Field Pennycress; pennycress) is being converted into a winter-annual oilseed crop that confers cover crop benefits when grown throughout the 12 million-hectares U.S. Midwest. To ensure a fit with downstream market demand, conversion involves not only improvements in yield and maturity through traditional breeding, but also improvements in the composition of the oil and protein through gene editing tools. The conversion process is similar to the path taken to convert rapeseed into Canola. In the case of field pennycress, the converted product that is suitable as a rotational crop is called CoverCress™ as marketed by CoverCress Inc. or golden pennycress if marketed by others. Off-season integration of a CoverCress crop into existing corn and soybean hectares would extend the growing season on established croplands and avoid displacement of food crops or ecosystems while yielding up to 1 billion liters of seed oil annually by 2030, with the potential to grow to 8 billion liters from production in the U.S. Midwest alone. The aviation sector is committed to carbon-neutral growth and reducing emissions of its global market, which in 2019 approached 122 billion liters of consumption in the U.S. and 454 billion liters globally. The oil derived from a CoverCress crop is ideally suited as a new bioenergy feedstock for the production of drop-in Sustainable Aviation Fuel (SAF), renewable diesel, biodiesel and other value-added coproducts. Through a combination of breeding and genomics-enabled mutagenesis approaches, considerable progress has been made in genetically improving yield and other agronomic traits. With USDA-NIFA funding and continued public and private investments, improvements to CoverCress germplasm and agronomic practices suggest that field-scale production can surpass 1,680 kg ha -1 (1,500 lb ac -1 ) in the near term. At current commodity prices, economic modeling predicts this level of production can be profitable across the entire supply chain. Two-thirds of the grain value is in oil converted to fuels and chemicals, and the other one-third is in the meal used as an animal feed, industrial applications, and potential plant-based protein products. In addition to strengthening rural communities by providing income to producers and agribusinesses, cultivating a CoverCress crop potentially offers a myriad of ecosystem services. The most notable service is water quality protection through reduced nutrient leaching and reduced soil erosion. Biodiversity enhancement by supporting pollinators’ health is also a benefit. While the efforts described herein are focused on the U.S., cultivation of a CoverCress crop will likely have a broader application to regions around the world with similar agronomic and environmental conditions.

09 BIOMASS FUELS↗
DOE OSTI2020

Multiplexed Knockouts in the Model Diatom Phaeodactylum by Episomal Delivery of a Selectable Cas9

Marine diatoms are eukaryotic microalgae that play significant ecological and biogeochemical roles in oceans. They also have significant potential as organismal platforms for exploitation to address biotechnological and industrial goals. In order to address both modes of research, sophisticated molecular and genetic tools are required. We presented here new and improved methodologies for introducing CRISPRCas9 to the model diatom Phaeodactylum tricornutum cells and a streamlined protocol for genotyping mutant cell lines with previously unknown phenotypes. First, bacterialconjugation was optimized for the delivery of Cas9 by transcriptionally fusing Cas9 to a selectable marker by the 2A peptide. An episome cloning strategy using both negative and positive selection was developed to streamline CRISPR-episome assembly. Next, cell line picking and genotyping strategies, that utilize manual sequencing curation, TIDE sequencing analysis, and a T7 endonuclease assay, were developed to shorten the time required to generate mutants. Following this new experimental pipeline, both singlegene and two-gene knockout cell lines were generated at mutagenesis efficiencies of 48% and 25%, respectively. Lastly, a protocol for precise gene insertions via CRISPRCas9 targeting was developed using particle-bombardment transformation methods. Overall, the novel Cas9 episome design and improved genotyping methods presented here allow for quick and easy genotyping and isolation of Phaeodactylum mutant cell lines (less than 3 weeks) without relying on a known phenotype to screen for mutants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Stable binding to phosphatidylserine-containing membranes requires conserved arginine residues in tandem C domains of blood coagulation factor VIII

At sites of vascular damage, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated platelet surfaces with activated factor IX (fIXa) to form the intrinsic “tenase” complex. Previous structural and mutational studies of fVIII have identified the C1 and C2 domains in binding to negatively charged membrane surfaces through β-hairpin loops with solvent-exposed hydrophobic residues and a ring of positively charged basic residues. Several hemophilia A-associated mutations within the C domains are suggested to disrupt lipid binding, preventing formation of the intrinsic tenase complex. In this study, we devised a novel platform for generating recombinant C1, C2, and C1C2 domain constructs and performed mutagenesis of several charged residues proximal to the putative membrane binding region of each C domain. Binding measurements between phosphatidylserine (PS)-containing lipid membrane surfaces and fVIII C domains demonstrated an ionic strength dependence on membrane binding affinity. Mutations to basic residues adjacent to the surface-exposed hydrophobic regions of C1 and C2 differentially disrupted membrane binding, with abrogation of binding occurring for mutations to conserved arginine residues in the C1 (R2163) and C2 (R2320) domains. Lastly, we determined the X-ray crystal structure of the porcine fVIII C2 domain bound to o-phospho-L-serine, the polar headgroup of PS, which binds to a basic cleft and makes charge-charge contact with R2320. We conclude that basic clefts in the fVIII C domains bind to PS-containing membranes through conserved arginine residues via a C domain modularity, where each C domain possesses modest electrostatic-dependent affinity and tandem C domains are required for high affinity binding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Dual incorporation of non-canonical amino acids enables production of post-translationally modified selenoproteins

Post-translational modifications (PTMs) can occur on almost all amino acids in eukaryotes as a key mechanism for regulating protein function. The ability to study the role of these modifications in various biological processes requires techniques to modify proteins site-specifically. One strategy for this is genetic code expansion (GCE) in bacteria. The low frequency of post-translational modifications in bacteria makes it a preferred host to study whether the presence of a post-translational modification influences a protein’s function. Genetic code expansion employs orthogonal translation systems engineered to incorporate a modified amino acid at a designated protein position. Selenoproteins, proteins containing selenocysteine, are also known to be post-translationally modified. Selenoproteins have essential roles in oxidative stress, immune response, cell maintenance, and skeletal muscle regeneration. Their complicated biosynthesis mechanism has been a hurdle in our understanding of selenoprotein functions. As technologies for selenocysteine insertion have recently improved, we wanted to create a genetic system that would allow the study of post-translational modifications in selenoproteins. By combining genetic code expansion techniques and selenocysteine insertion technologies, we were able to recode stop codons for insertion of N ε -acetyl- l -lysine and selenocysteine, respectively, into multiple proteins. The specificity of these amino acids for their assigned position and the simplicity of reverting the modified amino acid via mutagenesis of the codon sequence demonstrates the capacity of this method to study selenoproteins and the role of their post-translational modifications. Moreover, the evidence that Sec insertion technology can be combined with genetic code expansion tools further expands the chemical biology applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

CRISPR/Cas9-Induced fad2 and rod1 Mutations Stacked With f ae1 Confer High Oleic Acid Seed Oil in Pennycress (Thlaspi arvense L.)

Pennycress (Thlaspi arvense L.) is being domesticated as an oilseed cash cover crop to be grown in the off-season throughout temperate regions of the world. With its diploid genome and ease of directed mutagenesis using molecular approaches, pennycress seed oil composition can be rapidly tailored for a plethora of food, feed, oleochemical and fuel uses. Here, we utilized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to produce knockout mutations in the FATTY ACID DESATURASE2 (FAD2) and REDUCED OLEATE DESATURATION1 (ROD1) genes to increase oleic acid content. High oleic acid (18:1) oil is valued for its oxidative stability that is superior to the polyunsaturated fatty acids (PUFAs) linoleic (18:2) and linolenic (18:3), and better cold flow properties than the very long chain fatty acid (VLCFA) erucic (22:1). When combined with a FATTY ACID ELONGATION1 (fae1) knockout mutation, fad2 fae1 and rod1 fae1 double mutants produced ~90% and ~60% oleic acid in seed oil, respectively, with PUFAs in fad2 fae1 as well as fad2 single mutants reduced to less than 5%. MALDI-MS spatial imaging analyses of phosphatidylcholine (PC) and triacylglycerol (TAG) molecular species in wild-type pennycress embryo sections from mature seeds revealed that erucic acid is highly enriched in cotyledons which serve as storage organs, suggestive of a role in providing energy for the germinating seedling. In contrast, PUFA-containing TAGs are enriched in the embryonic axis, which may be utilized for cellular membrane expansion during seed germination and seedling emergence. Under standard growth chamber conditions, rod1 fae1 plants grew like wild type whereas fad2 single and fad2 fae1 double mutant plants exhibited delayed growth and overall reduced heights and seed yields, suggesting that reducing PUFAs below a threshold in pennycress had negative physiological effects. Taken together, our results suggest that combinatorial knockout of ROD1 and FAE1 may be a viable route to commercially increase oleic acid content in pennycress seed oil whereas mutations in FAD2 will likely require at least partial function to avoid fitness trade-offs.

09 BIOMASS FUELS↗
DOE OSTI2021

Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis

Monoterpenoids are the main components of plant essential oils and the active components of some traditional Chinese medicinal herbs like Mentha haplocalyx Briq ., Nepeta tenuifolia Briq ., Perilla frutescens (L.) Britt and Pogostemin cablin (Blanco) Benth. Pulegone reductase is the key enzyme in the biosynthesis of menthol and is required for the stereoselective reduction of the Δ 2,8 double bond of pulegone to produce the major intermediate menthone, thus determining the stereochemistry of menthol. However, the structural basis and mechanism underlying the stereoselectivity of pulegone reductase remain poorly understood. In this study, we characterized a novel (-)-pulegone reductase from Nepeta tenuifolia ( Nt PR), which can catalyze (-)-pulegone to (+)-menthone and (-)-isomenthone through our RNA-seq, bioinformatic analysis in combination with in vitro enzyme activity assay, and determined the structure of (+)-pulegone reductase from ( Mp PR) by using X-ray crystallography, molecular modeling and docking, site-directed mutagenesis, molecular dynamics simulations, and biochemical analysis. We identified and validated the critical residues in the crystal structure of Mp PR involved in the binding of the substrate pulegone. We also further identified that residues Leu56, Val282, and Val284 determine the stereoselectivity of the substrate pulegone, and mainly contributes to the product stereoselectivity. This work not only provides a starting point for the understanding of stereoselectivity of pulegone reductases, but also offers a basis for the engineering of menthone/menthol biosynthetic enzymes to achieve high-titer, industrial-scale production of enantiomerically pure products.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2020

Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal Antibodies

We report the novel crystal structure and characterization of symmetrical, homodimeric humanized heavy-chain-only antibodies or dimers (HC2s). HC2s were found to be significantly coexpressed and secreted along with mAbs from transient CHO HC/LC cotransfection, resulting in an unacceptable mAb developability attribute. Expression of full-length HC2s in the absence of LC followed by purification resulted in HC2s with high purity and thermal stability similar to conventional mAbs. The V H and C H 1 portion of the heavy chain (or Fd) was also efficiently expressed and yielded a stable, covalent, and reducible dimer (Fd2). Mutagenesis of all heavy chain cysteines involved in disulfide bond formation revealed that Fd2 intermolecular disulfide formation was similar to Fabs and elucidated requirements for Fd2 folding and expression. For one HC2, we solved the crystal structure of the Fd2 domain to 2.9 Å, revealing a highly symmetrical homodimer that is structurally similar to Fabs and is mediated by conserved (C H 1) and variable (V H ) contacts with all CDRs positioned outward for target binding. Interfacial dimer contacts revealed by the crystal structure were mutated for two HC2s and were found to dramatically affect HC2 formation while maintaining mAb bioactivity, offering a potential means to modulate novel HC2 formation through engineering. These findings indicate that human heavy-chain dimers can be secreted efficiently in the absence of light chains, may show good physicochemical properties and stability, are structurally similar to Fabs, offer insights into their mechanism of formation, and may be amenable as a novel therapeutic modality.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

The Ephb2 Receptor Uses Homotypic, Head-to-Tail Interactions within Its Ectodomain as an Autoinhibitory Control Mechanism

The Eph receptor tyrosine kinases and their ephrin ligands direct axon pathfinding and neuronal cell migration, as well as mediate many other cell–cell communication events. Their dysfunctional signaling has been shown to lead to various diseases, including cancer. The Ephs and ephrins both localize to the plasma membrane and, upon cell–cell contact, form extensive signaling assemblies at the contact sites. The Ephs and the ephrins are divided into A and B subclasses based on their sequence conservation and affinities for each other. The molecular details of Eph–ephrin recognition have been previously revealed and it has been documented that ephrin binding induces higher-order Eph assemblies, which are essential for full biological activity, via multiple, distinct Eph–Eph interfaces. One Eph–Eph interface type is characterized by a homotypic, head-to-tail interaction between the ligand-binding and the fibronectin domains of two adjacent Eph molecules. While the previous Eph ectodomain structural studies were focused on A class receptors, we now report the crystal structure of the full ectodomain of EphB2, revealing distinct and unique head-to-tail receptor–receptor interactions. The EphB2 structure and structure-based mutagenesis document that EphB2 uses the head-to-tail interactions as a novel autoinhibitory control mechanism for regulating downstream signaling and that these interactions can be modulated by posttranslational modifications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Crystal Structure of CYP3A4 Complexed with Fluorol Identifies the Substrate Access Channel as a High-Affinity Ligand Binding Site

Cytochrome P450 3A4 (CYP3A4) is a major human drug-metabolizing enzyme, notoriously known for its extreme substrate promiscuity, allosteric behavior, and implications in drug–drug interactions. Despite extensive investigations, the mechanism of ligand binding to CYP3A4 is not fully understood. We determined the crystal structure of CYP3A4 complexed with fluorol, a small fluorescent dye that can undergo hydroxylation. In the structure, fluorol associates to the substrate channel, well suited for the binding of planar polyaromatic molecules bearing polar groups, through which stabilizing H-bonds with the polar channel residues, such as Thr224 and Arg372, can be established. Mutagenesis, spectral, kinetic, and functional data confirmed the involvement but not strict requirement of Thr224 for the association of fluorol. Collectively, our data identify the substrate channel as a high-affinity ligand binding site and support the notion that hydrophobic ligands first dock to the nearby peripheral surface, before migrating to the channel and, subsequently, into the active site.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural Similarities and Overlapping Activities among Dihydroflavonol 4-Reductase, Flavanone 4-Reductase, and Anthocyanidin Reductase Offer Metabolic Flexibility in the Flavonoid Pathway

Flavonoids are potent antioxidants that play a role in defense against pathogens, UV-radiation, and the detoxification of reactive oxygen species. Dihydroflavonol 4-reductase (DFR) and flavanone 4-reductase (FNR) reduce dihydroflavonols and flavanones, respectively, using NAD(P)H to produce flavan-(3)-4-(di)ols in flavonoid biosynthesis. Anthocyanidin reductase (ANR) reduces anthocyanidins to flavan-3-ols. In addition to their sequences, the 3D structures of recombinant DFR, FNR and ANR from sorghum and switchgrass showed a high level of similarity. The catalytic mechanism, substrate-specificity and key residues of three reductases were deduced from crystal structures, site-directed mutagenesis, molecular docking, kinetics, and thermodynamic ana-lyses. Although DFR displayed its highest activity against dihydroflavonols, it also showed activity against flavanones and anthocyanidins. It was inhibited by the flavonol quercetin and high concentrations of dihydroflavonols/flavonones. SbFNR1 and SbFNR2 did not show any activity against dihydroflavonols. However, SbFNR1 displayed activity against flavanones and ANR activity against two anthocyanidins, cyanidin and pelargonidin. Therefore, SbFNR1 and SbFNR2 could be specific ANR isozymes without delphinidin activity. Sorghum has high concentrations of 3-deoxyanthocyanidins in vivo, supporting the observed high activity of SbDFR against flavonols. Mining of expression data indicated substantial induction of these three reductase genes in both switchgrass and sorghum in response to biotic stress. Key signature sequences for proper DFR/ANR classification are proposed and could form the basis for future metabolic engineering of flavonoid metabolism.

3-deoxyanthocyanidin↗
DOE OSTI2020

Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study

Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today’s agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized more quickly than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell’s growth rate to the target gene’s function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.

59 BASIC BIOLOGICAL SCIENCES↗