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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 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Characterization of aromatic acid/proton symporters in Pseudomonas putida KT2440 toward efficient microbial conversion of lignin-related aromatics

Pseudomonas putida KT2440 (hereafter KT2440) is a well-studied platform bacterium for the production of industrially valuable chemicals from heterogeneous mixtures of aromatic compounds obtained from lignin depolymerization. KT2440 can grow on lignin-related monomers, such as ferulate (FA), 4-coumarate (4CA), vanillate (VA), 4-hydroxybenzoate (4HBA), and protocatechuate (PCA). Genes associated with their catabolism are known, but knowledge about the uptake systems remains limited. In this work, we studied the KT2440 transporters of lignin-related monomers and their substrate selectivity. Based on the inhibition by protonophores, we focused on five genes encoding aromatic acid/H+ symporter family transporters categorized into major facilitator superfamily that uses the proton motive force. Furthermore, the mutants of PP_1376 (pcaK) and PP_3349 (hcnK) exhibited significantly reduced growth on PCA/4HBA and FA/4CA, respectively, while no change was observed on VA for any of the five gene mutants. At pH 9.0, the conversion of these compounds by hcnK mutant (FA/4CA) and vanK mutant (VA) was dramatically reduced, revealing that these transporters are crucial for the uptake of the anionic substrates at high pH. Uptake assays using 14 C-labeled substrates in Escherichia coli and biosensor-based assays confirmed that PcaK, HcnK, and VanK have ability to take up PCA, FA/4CA, and VA/PCA, respectively. Additionally, analyses of the predicted protein structures suggest that the size and hydropathic properties of the substrate-binding sites of these transporters determine their substrate preferences. Overall, this study reveals that at physiological pH, PcaK and HcnK have a major role in the uptake of PCA/4HBA and FA/4CA, respectively, and VanK is a VA/PCA transporter. This information can contribute to the engineering of strains for the efficient conversion of lignin-related monomers to value-added chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Human N -acetylglucosaminyltransferase II substrate recognition uses a modular architecture that includes a convergent exosite

Asn-linked oligosaccharides are extensively modified during transit through the secretory pathway, first by trimming of the nascent glycan chains and subsequently by initiating and extending multiple oligosaccharide branches from the trimannosyl glycan core. Trimming and branching pathway steps are highly ordered and hierarchal based on the precise substrate specificities of the individual biosynthetic enzymes. A key committed step in the synthesis of complex-type glycans is catalyzed by N-acetylglucosaminyltransferase II (MGAT2), an enzyme that generates the second GlcNAcβ1,2- branch from the trimannosyl glycan core using UDP-GlcNAc as the sugar donor. We determined the structure of human MGAT2 as a Mn 2+ -UDP donor analog complex and as a GlcNAcMan 3 GlcNAc 2 -Asn acceptor complex to reveal the structural basis for substrate recognition and catalysis. The enzyme exhibits a GT-A Rossmann-like fold that employs conserved divalent cation-dependent substrate interactions with the UDP-GlcNAc donor. MGAT2 interactions with the extended glycan acceptor are distinct from other related glycosyltransferases. These interactions are composed of a catalytic subsite that binds the Man-α1,6- monosaccharide acceptor and a distal exosite pocket that binds the GlcNAc-β1,2Man-α1,3Manβ- substrate “recognition arm.” Recognition arm interactions are similar to the enzyme–substrate interactions for Golgi α-mannosidase II, a glycoside hydrolase that acts just before MGAT2 in the Asn-linked glycan biosynthetic pathway. Here, these data suggest that substrate binding by MGAT2 employs both conserved and convergent catalytic subsite modules to provide substrate selectivity and catalysis. More broadly, the MGAT2 active-site architecture demonstrates how glycosyltransferases create complementary modular templates for regiospecific extension of glycan structures in mammalian cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structure-property relationships and mobility optimization in sputtered La-doped BaSn O 3 films: Toward 100cm 2 V -1 s -1

he wide band gap semiconducting perovskite BaSnO 3 is of high current interest due to outstanding room temperature mobility at high electron density, fueled by potential applications in oxide, transparent, and power electronics. Due in part to a lack of lattice-matched substrates, BaSnO 3 thin films suffer from high defect densities, however, limiting electron mobility. Additionally, the vast majority of BaSnO 3 thin film research has focused on pulsed laser deposition or molecular beam epitaxy. In this work, we present an exhaustive optimization of the mobility of Ba 0.98 La 0.02 SnO 3 films grown by a scalable, high-throughput method: high-pressure-oxygen sputter deposition. Considering target synthesis conditions, substrate selection, buffer layer structure, deposition temperature, deposition rate, thickness, and postdeposition annealing conditions, and by combining high-resolution x-ray diffraction, reciprocal space mapping, rocking curve analysis, scanning transmission electron microscopy, atomic force microscopy, and temperature-dependent electronic transport measurements, detailed understanding of synthesis-structure-property relationships is attained. Optimized room temperature mobility of 96 cm 2 V –1 s –1 is achieved in vacuum-annealed GdScO 3 (110)/BaSnO 3 (120 nm)/Ba 0.98 La 0.02 SnO 3 (200 nm) heterostructures, as well as 92 cm 2 V –1 s –1 on unbuffered substrates and 87 2 V –1 s –1 without postdeposition annealing. These results, including important trends in defect densities and a surprising dependence of mobility on lattice mismatch, substantially expand the understanding of the interplay between deposition conditions, microstructure, and transport in doped BaSnO 3 films, establishing competitive mobilities in films fabricated via a scalable, high-throughput, industry-standard technique.

36 MATERIALS SCIENCE↗
DOE OSTI2022

Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

Abstract Sequence variation in related proteins is an important characteristic that modulates activity and selectivity. An example of a protein family with a large degree of sequence variation is that of bacterial sortases, which are cysteine transpeptidases on the surface of gram‐positive bacteria. Class A sortases are responsible for attachment of diverse proteins to the cell wall to facilitate environmental adaption and interaction. These enzymes are also used in protein engineering applications for sortase‐mediated ligations (SML) or sortagging of protein targets. We previously investigated SrtA from Streptococcus pneumoniae , identifying a number of putative β7–β8 loop‐mediated interactions that affected in vitro enzyme function. We identified residues that contributed to the ability of S. pneumoniae SrtA to recognize several amino acids at the P1′ position of the substrate motif, underlined in LPXT G , in contrast to the strict P1′ Gly recognition of SrtA from Staphylococcus aureus . However, motivated by the lack of a structural model for the active, monomeric form of S. pneumoniae SrtA, here, we expanded our studies to other Streptococcus SrtA proteins. We solved the first monomeric structure of S. agalactiae SrtA which includes the C‐terminus, and three others of β7–β8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified β7–β8 loop‐mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. A greater understanding of individual SrtA sequence and structural determinants of target selectivity may also facilitate the design or discovery of improved sortagging tools.

Gao, Melody↗
NASA NTRS2004

High Sensitivity, Low Power Nano Sensors and Devices for Chemical Sensing

The chemical sensor market has been projected to grow to better than $40 billion dollars worldwide within the next 10 years. Some of the primary motivations to develop nanostructured chemical sensors are monitoring and control of environmental pollution; improved diagnostics for consumption; improvement in measurement precision and accuracy; and improved detection limits for Homeland security, battlefield environments, and process and quality control of industrial applications. In each of these applications, there is demand for sensitivity, selectivity and stability of environmental and biohazard detection and capture beyond what is currently commercially available. Nanotechnology offers the ability to work at the molecular level, atom by atom, to create large structures with fundamentally new molecular organization. It is essentially concerned with materials, devices, and systems whose structures and components exhibit novel and significantly improved physical, chemical and biological properties, phenomena, and process control due to their nanoscale size. One such nanotechnology-enabled chemical sensor has been developed at NASA Ames leveraging nanostructures, such as single walled carbon nanotubes (SWNTs) and metal oxide nanobelts or nanowires, as a sensing medium bridging a pair of interdigitated electrodes (IDE) realized through a silicon-based microfabrication and micromachining technique. The DE fingers are fabricated on a silicon substrate using standard photolithography and thin film metallization techniques. It is noteworthy that the fabrication techniques employed are not confined to the silicon substrate. Through spin casting and careful substrate selection (i.e. clothing, glass, polymer, etc.), additional degrees of freedom can be exploited to enhance sensitivity or to conform to unique applications. Both in-situ growth of nanostructured materials and casting of nanostructured dispersions were used to produce analogous chemical sensing devices.

Li, Jing↗
DOE OSTI2019

Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents

Cellulases from glycoside hydrolase family 5 (GH5) are key endoglucanase enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical (βα) 8 -barrel structure, where each (βα) module is essential for the enzyme’s stability and activity. Despite their shared topology, the thermostability of GH5 endoglucanase enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on the previously characterized thermophilic GH5 endoglucanase Egl5A from Talaromyces emersonii ( Te Egl5A), which has an optimal temperature of 90°C, we created 10 hybrid enzymes with elements of the mesophilic endoglucanase Cel5 from Stegonsporium opalus ( So Cel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optimum (10 and 20°C), the temperature at which the protein lost 50% of its activity ( T 50 ) (15 and 19°C), and the melting temperature ( T m ) (16.5 and 22.9°C) and extended half-lives ( t 1/2 ) (~240- and 650-fold at 55°C) relative to the values for the mesophilic parent enzyme and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 endoglucanase, Cel5 from Aspergillus niger ( An Cel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics (MD) simulations of both the So Cel5 and Te Egl5A parent enzymes and their hybrids, we hypothesize that improved hydrophobic packing of the interface between α 2 and α 3 is the primary mechanism by which the hybrid enzymes increase their thermostability relative to that of the mesophilic parent So Cel5.Thermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM barrel fragments, and even fragments from unrelated folds, to generate new structures. However, little research has been done on GH5 endoglucanases. In this study, two GH5 endoglucanases exhibiting TIM barrel structure, So Cel5 and Te Egl5A, with different thermal properties, were hybridized to study the roles of different (βα) motifs. This work illustrates the role that structure-guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.

09 BIOMASS FUELS↗
DOE OSTI2021

Discovery of an ultraspecific triuret hydrolase (TrtA) establishes the triuret biodegradation pathway

Triuret (carbonyldiurea) is an impurity found in industrial urea fertilizer (<0.1% w/w) that is applied, worldwide, around 300 million pounds each year on agricultural lands. In addition to anthropogenic sources, endogenous triuret has been identified in amoeba and human urine, the latter being diagnostic for hypokalemia. The present study is the first to describe the metabolic breakdown of triuret, which funnels into biuret metabolism. We identified the gene responsible for triuret decomposition ( trtA ) in bacterial genomes, clustered with biuH , which encodes biuret hydrolase and has close protein sequence homology. TrtA is a member of the isochorismatase-like hydrolase (IHL) protein family, similarly to BiuH, and has a catalytic efficiency (k cat/ K M ) of 6 x 10 5 M –1 s –1 , a K M for triuret of 20 μM, and exquisite substrate specificity. Indeed, TrtA has four orders of magnitude less activity with biuret. Crystal structures of TrtA in apo and holo form were solved and compared with the BiuH structure. The high substrate selectivity was found to be conveyed by second shell residues around each active site. Mutagenesis of residues conserved in TrtA to the alternate consensus found in BiuHs revealed residues critical to triuret hydrolase activity but no single mutant evolved more biuret activity, and likely a combination of mutations is required to interconvert between TrtA, BiuH functions. TrtA-mediated triuret metabolism is relatively rare in recorded genomes (1–2%), but is largely found in plant-associated, nodulating, and endophytic bacteria. This study suggests functions for triuret hydrolase in certain eukaryotic intermediary processes and prokaryotic intermediary or biodegradative metabolism.

36 MATERIALS SCIENCE↗
DOE OSTI2019

Directed Evolution Reveals the Functional Sequence Space of an Adenylation Domain Specificity Code

Nonribosomal peptides are important natural products biosynthesized by nonribosomal peptide synthetases (NRPSs). Adenylation (A) domains of NRPSs are highly specific for the substrate they recognize. This recognition is determined by 10 residues in the substrate-binding pocket, termed the specificity code. This finding led to the proposal that nonribosomal peptides could be altered by specificity code swapping. Unfortunately, this approach has proven, with few exceptions, to be unproductive; changing the specificity code typically results in broadened specificity or poor function. To enhance our understanding of A domain substrate selectivity, we carried out a detailed analysis of the specificity code from the A domain of EntF, an NRPS involved in enterobactin biosynthesis in Escherichia coli. Using directed evolution and a genetic selection, we determined which sites in the code have strict residue requirements and which are tolerant of variation. We showed that the EntF A domain, and other l-Ser-specific A domains, have a functional sequence space for l-Ser recognition, rather than a single code. This functional space is more expansive than the aggregate of all characterized l-Ser-specific A domains: we identified 152 new l-Ser specificity codes. Together, our data provide essential insights into how to overcome the barriers that prevent rational changes to A domain specificity.

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

Comparative performance of PETase as a function of reaction conditions, substrate properties, and product accumulation

There is keen interest to develop new technologies to recycle the plastic poly(ethylene terephthalate) (PET). To this end, the use of PET-hydrolyzing enzymes has shown promise for PET deconstruction to its monomers, terephthalate (TPA) and ethylene glycol (EG). Here, we compare the Ideonella sakaiensis PETase wild-type enzyme to a previously reported improved variant (W159H/S238F). We compare the thermostability of each enzyme and describe a 1.45 Å resolution structure of the mutant, highlighting changes in the substrate binding cleft compared to the wild-type enzyme. Subsequently, the performance of the wild-type and variant enzyme was compared as a function of temperature, substrate morphology, and reaction mixture composition. These studies show that reaction temperature has the strongest influence on performance between the two enzymes. We also show that both enzymes achieve higher levels of PET conversion for substrates with moderate crystallinity relative to amorphous substrates. Finally, we assess the impact of product accumulation on reaction progress for the hydrolysis of both PET and bis(2-hydroxyethyl) terephthalate (BHET). Each enzyme displays different inhibition profiles to mono(2-hydroxyethyl) terephthalate (MHET) and TPA, while both are sensitive to inhibition by EG. Overall, this work highlights the importance of reaction conditions, substrate selection, and product accumulation for catalytic performance of PET-hydrolyzing enzymes, which have implications for enzyme screening in the development of enzyme-based polyester recycling.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Evidence that cytochrome b{sub 5} acts as a redox donor in CYP17A1 mediated androgen synthesis

Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second C−C lyase step, at the expense of glucocorticoid production. Cytochrome b{sub 5} (cyt b{sub 5}) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b{sub 5} increases androgen biosynthesis. Cyt b{sub 5} could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b{sub 5} could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b{sub 5}, we generated a redox-inactive form of cyt b{sub 5}, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b{sub 5}), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b{sub 5} were enhanced ∼5 fold as compared to reactions in the absence of cyt b{sub 5}. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b{sub 5}. Upon addition of Mn-b{sub 5} to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b{sub 5}-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal Fe−S vibrational frequency. Thus, although Mn-b{sub 5} binds to CYP17A1, it is unable to enhance the lyase reaction, strongly suggesting that cyt b{sub 5} has a redox effector role in enhancement of the CYP17A1 mediated lyase reaction necessary for androgen synthesis. - Highlights: • Cyt b{sub 5} role in human CYP17A1 mediated androgen synthesis was probed in Nanodiscs. • Native cyt b{sub 5} enhances androgen synthesis by CYP17A1. • Redox inactive Mn cyt b{sub 5} does not enhance androgen synthesis by CYP17A1. • Interactions with Cyt b{sub 5} perturb Fe−S and heme Raman modes of CYP17A1. • Cyt b{sub 5} acts as a redox donor for CYP17A1 mediated androgen synthesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Cullin 3 regulates ADAMs-mediated ectodomain shedding of amphiregulin

Highlights: • ADAMs-mediated EGF family shedding is regulated by actin cytoskeleton. • Cul3 regulates AREG shedding by modulating actin cytoskeleton. • The neddylation is crucial step for Cul3 to regulate ectodomain shedding. • Cul3 E3 ubiquitin ligase complex is promising target to regulate ADAM activity. A disintegrin and metalloproteinase (ADAM) family are crucial enzymes for ectodomain shedding of multiple substrates and are involved in diverse biologic and pathologic processes. However, the molecular mechanism underlying substrate selectivity of ADAMs is poorly understood. In this study, we observed that disruption of actin polymerization by pharmacological inhibitors, latrunculin A (LatA) and cytochalasin D (CyD), induced ectodomain shedding of epidermal growth factor (EGF) family ligands. Induced shedding activity by LatA or CyD was suppressed by a metalloprotease inhibitor KB-R7785, indicating that ADAMs-mediated shedding is tightly controlled by actin cytoskeleton. We also investigated roles of cullin family, a component of cullin-RING based E3 ubiquitin ligases, in ectodomain shedding, since cullin family is implicated in the regulation of cytoskeletal dynamics. Knockdown of cullin 3 (Cul3) by a specific siRNA inhibited ectodomain shedding of amphiregulin (AREG), a member of EGF family, and responses were associated with activation of RhoA GTPase and induction of stress fiber formation. On the other hand, the RhoA inhibitor C3 transferase rescued AREG shedding reduced by Cul3 knockdown. These results describe a novel molecular mechanism of Cul3 to regulate AREG shedding by modulating cytoskeletal dynamics in a RhoA dependent manner.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Molecular basis for dual functions in pilus assembly modulated by the lid of a pilus-specific sortase

The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Self-Assembled Epitaxial Au–Oxide Vertically Aligned Nanocomposites for Nanoscale Metamaterials

Metamaterials made of nanoscale inclusions or artificial unit cells exhibit exotic optical properties that do not exist in natural materials. Promising applications, such as super-resolution imaging, cloaking, hyperbolic propagation, and ultrafast phase velocities have been demonstrated based on mostly micrometer-scale metamaterials and few nanoscale metamaterials. To date, most metamaterials are created using costly and tedious fabrication techniques with limited paths toward reliable large-scale fabrication. In this work, we demonstrate the one-step direct growth of self-assembled epitaxial metal–oxide nanocomposites as a drastically different approach to fabricating large-area nanostructured metamaterials. Using pulsed laser deposition, we fabricated nanocomposite films with vertically aligned gold (Au) nanopillars (~20 nm in diameter) embedded in various oxide matrices with high epitaxial quality. Strong, broad absorption features in the measured absorbance spectrum are clear signatures of plasmon resonances of Au nanopillars. By tuning their densities on selected substrates, anisotropic optical properties are demonstrated via angular dependent and polarization resolved reflectivity measurements and reproduced by full-wave simulations and effective medium theory. Our model predicts exotic properties, such as zero permittivity responses and topological transitions. In conclusion, our studies suggest that these self-assembled metal–oxide nanostructures provide an exciting new material platform to control and enhance optical response at nanometer scales.

36 MATERIALS SCIENCE↗
DOE OSTI2019

How the Complementarity at Vicinal Steps Enables Growth of 2D Monocrystals

Large 2D monocrystals are highly sought after yet hard to achieve; unlike graphene, most dichalcogenides and h-BN possess low symmetry, which allows for nucleation of mutually inverted pieces, merging into polycrystals replete with grain boundaries. On vicinal substrate surfaces such growing pieces were observed to orient alike, and very recently this effect apparently enabled the growth of large single crystal h-BN. Addressing the compelling questions of how such a growth process can operate and what the key mechanisms are is crucial in guiding the substrate selection for optimal synthesis of perhaps many materials. To this end, the basic crystallography and atomistic-modeling theory presented here reveal (i) how the undulations of the ever-wandering steps do not, surprisingly, disturb the orientations of the attached 2D-nuclei, whose direction remains robust owing to complementarity between the meandering step and h-BN counterpart if their kinks have similar size of negligible misfit, δk < 0.1 Å. (ii) Stronger chemical affinity of metal to the N atoms at the zigzag edge of h-BN singles out its particular orientation, without evidence of any epitaxy, at the edge or to the surface. (iii) The monocrystal integrity requires unhindered growth spillover across the steps and the seamless healing of the residual fissures, caused by the very same steps necessary for co-orientation. Molecular dynamics simulations show this happening for the steps not taller than the BN bond, s < 1.44 Å. Furthermore, these criteria point to [–1 1 2] steps on the Cu (110) surface, in accord with experimental results, while other possibilities can also be predicted.

2D material↗
DOE OSTI2022

Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity

Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5–9.0 and temperatures from 30–70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2017

Non-specific activities of the major herbicide-resistance gene BAR

Bialaphos resistance (BAR) and phosphinothricin acetyltransferase (PAT) genes, which convey resistance to the broad-spectrum herbicide phosphinothricin (also known as glufosinate) via N-acetylation, have been globally used in basic plant research and genetically engineered crops. Although early in vitro enzyme assays showed that recombinant BAR and PAT exhibit substrate preference toward phosphinothricin over the 20 proteinogenic amino acids, indirect effects of BAR-containing transgenes in planta, including modified amino acid levels, have been seen but without the identification of their direct causes. Combining metabolomics, plant genetics and biochemical approaches, we show that transgenic BAR indeed converts two plant endogenous amino acids, aminoadipate and tryptophan, to their respective N-acetylated products in several plant species. We report the crystal structures of BAR, and further delineate structural basis for its substrate selectivity and catalytic mechanism. Through structure-guided protein engineering, we generated several BAR variants that display significantly reduced non-specific activities compared with its wild-type counterpart in vivo. Here, the transgenic expression of enzymes can result in unintended off-target metabolism arising from enzyme promiscuity. Understanding such phenomena at the mechanistic level can facilitate the design of maximally insulated systems featuring heterologously expressed enzymes.

59 BASIC BIOLOGICAL SCIENCES↗