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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 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Characterization of alternate encounter assemblies of SARS-CoV-2 main protease

The assembly of two monomeric constructs spanning segments 1-199 (MPro 1-199 ) and 10-306 (MPro 10-306 ) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ~7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro 1-199 and MPro 10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201 to 214 onto a second helical domain of MPro 10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro 1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro 10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ~6 μM MPro 1-199 . Catalytic activity arises primarily from MPro 1-199 and is dependent on the interface interactions involving the N-finger residues 1 to 9 of MPro 1-199 and E290 of MPro 10-306 . Importantly, our results confirm that a single N-finger region with its associated intersubunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Bivalent recognition of fatty acyl-CoA by a human integral membrane palmitoyltransferase

S-acylation, also known as palmitoylation, is the most abundant form of protein lipidation in humans. This reversible posttranslational modification, which targets thousands of proteins, is catalyzed by 23 members of the DHHC family of integral membrane enzymes. DHHC enzymes use fatty acyl-CoA as the ubiquitous fatty acyl donor and become autoacylated at a catalytic cysteine; this intermediate subsequently transfers the fatty acyl group to a cysteine in the target protein. Protein S-acylation intersects with almost all areas of human physiology, and several DHHC enzymes are considered as possible therapeutic targets against diseases such as cancer. These efforts would greatly benefit from a detailed understanding of the molecular basis for this crucial enzymatic reaction. Here, in this study, we combine X-ray crystallography with all-atom molecular dynamics simulations to elucidate the structure of the precatalytic complex of human DHHC20 in complex with palmitoyl CoA. The resulting structure reveals that the fatty acyl chain inserts into a hydrophobic pocket within the transmembrane spanning region of the protein, whereas the CoA headgroup is recognized by the cytosolic domain through polar and ionic interactions. Biochemical experiments corroborate the predictions from our structural model. We show, using both computational and experimental analyses, that palmitoyl CoA acts as a bivalent ligand where the interaction of the DHHC enzyme with both the fatty acyl chain and the CoA headgroup is important for catalytic chemistry to proceed. This bivalency explains how, in the presence of high concentrations of free CoA under physiological conditions, DHHC enzymes can efficiently use palmitoyl CoA as a substrate for autoacylation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

CO 2 -assisted ethane oxidative dehydrogenation over MoO x catalysts supported on reducible CeO 2 –TiO 2

Supported MoO x on mixed CeO 2 –TiO 2 was investigated for the oxidative dehydrogenation of ethane (ODHE) using CO 2 as a mild oxidant. Raman spectroscopic characterization of the synthesized catalysts under dehydrated conditions suggested that surface MoO x species prefer to anchor on the crystalline domains of TiO 2 . Upon increasing the amount of CeO 2 in the mixed oxide support, significant spectral changes were observed, especially in the ~900–950 cm –1 region where Mo–O–M bonds are expected. The catalytic behaviors of Mo as opposed to pure support materials were distinct. As the ceria content in the support increased, MoO x catalysts promoted oxidative dehydrogenation pathways via the Mars–van Krevelen mechanism, while pure supports appeared to favor ethane direct dehydrogenation. Investigation of structure–function relationships via in situ Raman spectroscopic efforts revealed that adding ceria not only changed the redox properties of the support but also improved those of the deposited amorphous MoO x species. We also show that upon incorporation of ceria into the support, CO 2 directly participates in the reoxidation of the dispersed MoO x species during catalysis. Here, this effect was distinct from the participation of CO 2 in the reverse water gas shift reaction. Operando Raman spectra revealed that the presence of CO 2 prolonged the existence of the 930 cm –1 feature which appears to correlate well with the relative contribution of the oxidative versus non-oxidative pathway in ethane dehydrogenation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1994

Multiblock analysis for Shuttle Orbiter reentry heating from Mach 24 to Mach 12

A multiblock, laminar heating analysis for the shuttle orbiter at three trajectory points ranging from Mach 24.3 to Mach 12.86 on reentry is described. The analysis is performed using the Langley Aerothermodynamic Upwind Relaxation Algorithm with a seven species chemical nonequilibrium model. A finite-catalytic-wall model appropriate for shuttle tiles at a radiative equilibrium wall temperature is applied. Computed heating levels are generally in good agreement with the flight data, although a few rather large discrepancies remain unexplained. The multiblock relaxation strategy partitions the flowfield into manageable blocks requiring a fraction of the computational resources (time and memory) required by a full domain approach. In fact, the computational cost for a solution at even a single trajectory point would be prohibitively expensive at the given resolution without the multiblock approach. Converged blocks are reassembled to enable a fully coupled converged solution over the entire vehicle, starting from a nearly converged initial condition.

LANGLEY AEROTHERMODYNAMIC UPWI↗
DOE OSTI2023

Novel viruses of the family Partitiviridae discovered in Saccharomyces cerevisiae

It has been 49 years since the last discovery of a new virus family in the model yeast Saccharomyces cerevisiae . A large-scale screen to determine the diversity of double-stranded RNA (dsRNA) viruses in S . cerevisiae has identified multiple novel viruses from the family Partitiviridae that have been previously shown to infect plants, fungi, protozoans, and insects. Most S . cerevisiae partitiviruses (ScPVs) are associated with strains of yeasts isolated from coffee and cacao beans. The presence of partitiviruses was confirmed by sequencing the viral dsRNAs and purifying and visualizing isometric, non-enveloped viral particles. ScPVs have a typical bipartite genome encoding an RNA-dependent RNA polymerase (RdRP) and a coat protein (CP). Phylogenetic analysis of ScPVs identified three species of ScPV, which are most closely related to viruses of the genus Cryspovirus from the mammalian pathogenic protozoan Cryptosporidium parvum . Molecular modeling of the ScPV RdRP revealed a conserved tertiary structure and catalytic site organization when compared to the RdRPs of the Picornaviridae . The ScPV CP is the smallest so far identified in the Partitiviridae and has structural homology with the CP of other partitiviruses but likely lacks a protrusion domain that is a conspicuous feature of other partitivirus particles. ScPVs were stably maintained during laboratory growth and were successfully transferred to haploid progeny after sporulation, which provides future opportunities to study partitivirus-host interactions using the powerful genetic tools available for the model organism S . cerevisiae .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The interaction between transport-segment DNA and topoisomerase IA—crystal structure of MtbTOP1 in complex with both G- and T-segments

Abstract Each catalytic cycle of type IA topoisomerases has been proposed to comprise multistep reactions. The capture of the transport-segment DNA (T-segment) into the central cavity of the N-terminal toroidal structure is an important action, which is preceded by transient gate-segment (G-segment) cleavage and succeeded by G-segment religation for the relaxation of negatively supercoiled DNA and decatenation of DNA. The T-segment passage in and out of the central cavity requires significant domain–domain rearrangements, including the movement of D3 relative to D1 and D4 for the opening and closing of the gate towards the central cavity. Here we report a direct observation of the interaction of a duplex DNA in the central cavity of a type IA topoisomerase and its associated domain–domain conformational changes in a crystal structure of a Mycobacterium tuberculosis topoisomerase I complex that also has a bound G-segment. The duplex DNA within the central cavity illustrates the non-sequence-specific interplay between the T-segment DNA and the enzyme. The rich structural information revealed from the novel topoisomerase–DNA complex, in combination with targeted mutagenesis studies, provides new insights into the mechanism of the topoisomerase IA catalytic cycle.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Hot Electron-Driven Photocatalysis Using Sub-5 nm Gap Plasmonic Nanofinger Arrays

Semiconductor photocatalysis has received increasing attention because of its potential to address problems related to the energy crisis and environmental issues. However, conventional semiconductor photocatalysts, such as TiO2 and ZnO, can only be activated by ultraviolet light due to their wide band gap. To extend the light absorption into the visible range, the localized surface plasmon resonance (LSPR) effect of noble metal nanoparticles (NPs) has been widely used. Noble metal NPs can couple incident visible light energy to strong LSPR, and the nonradiative decay of LSPR generates nonthermal hot carriers that can be injected into adjacent semiconductor material to enhance its photocatalytic activity. Here we demonstrate that nanoimprint-defined gap plasmonic nanofinger arrays can function as visible light-driven plasmonic photocatalysts. The sub-5 nm gaps between pairs of collapsed nanofingers can support ultra-strong plasmon resonance and thus boost the population of hot carriers. The semiconductor material is exactly placed at the hot spots, providing an efficient pathway for hot carrier injection from plasmonic metal to catalytic materials. This nanostructure thus exhibits high plasmon-enhanced photocatalytic activity under visible light. The hot carrier injection mechanism of this platform was systematically investigated. The plasmonic enhancement factor was calculated using the finite-difference time-domain (FDTD) method and was consistent with the measured improvement of the photocatalytic activity. This platform, benefiting from the precise controllable geometry, provides a deeper understanding of the mechanism of plasmonic photocatalysis.

42 ENGINEERING↗
DOE OSTI2021

A Novel Glutathione S-Transferase Gtt2 Class (VpGSTT2) Is Found in the Genome of the AHPND/EMS Vibrio parahaemolyticus Shrimp Pathogen

Glutathione S-transferases are a family of detoxifying enzymes that catalyze the conjugation of reduced glutathione (GSH) with different xenobiotic compounds using either Ser, Tyr, or Cys as a primary catalytic residue. We identified a novel GST in the genome of the shrimp pathogen V. parahaemolyticus FIM- S1708+, a bacterial strain associated with Acute Hepatopancreatic Necrosis Disease (AHPND)/Early Mortality Syndrome (EMS) in cultured shrimp. This new GST class was named Gtt2. It has an atypical catalytic mechanism in which a water molecule instead of Ser, Tyr, or Cys activates the sulfhydryl group of GSH. The biochemical properties of Gtt2 from Vibrio parahaemolyticus (VpGSTT2) were characterized using kinetic and crystallographic methods. Recombinant VpGSTT2 was enzymatically active using GSH and CDNB as substrates, with a specific activity of 5.7 units/mg. Low affinity for substrates was demonstrated using both Michaelis–Menten kinetics and isothermal titration calorimetry. The crystal structure showed a canonical two-domain structure comprising a glutathione binding G-domain and a hydrophobic ligand H domain. A water molecule was hydrogen-bonded to residues Thr9 and Ser 11, as reported for the yeast Gtt2, suggesting a primary role in the reaction. Molecular docking showed that GSH could bind at the G-site in the vicinity of Ser11. G-site mutationsT9A and S11A were analyzed. S11A retained 30% activity, while T9A/S11A showed no detectable activity. VpGSTT2 was the first bacterial Gtt2 characterized, in which residues Ser11 and Thr9 coordinated a water molecule as part of a catalytic mechanism that was characteristic of yeast GTT2. The GTT2 family has been shown to provide protection against metal toxicity; in some cases, excess heavy metals appear in shrimp ponds presenting AHPND/EMS. Further studies may address whether GTT2 in V. parahaemolyticus pathogenic strains may provide a competitive advantage as a novel detoxification mechanism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Single-Walled Zeolitic Nanotube–Poly(oxazoline) Nanocomposites as Heterogeneous Catalysts for Acid–Base Cascade Reactions

Zeolites with a unique, 1-dimensional form factor were recently discovered − zeolite nanotubes (ZNTs). Here we describe the synthesis and characterization of NaH-ZNTpoly( oxazoline) composites targeting liquid-phase acid−base cascade catalysis. NaH-ZNT, a one-dimensional zeolite analogue with mesoporosity (3−4 nm) associated with nanotubes and inherent Brønsted acid sites associated with the microporous zeolite domains, is functionalized with poly(oxazoline)-based triblock copolymers with varying molecular weights (3−17 kDa). The composites are characterized using N2 sorption, STEM, FTIR, and elemental analysis, confirming successful grafting and preservation of the zeolite nanotube structure. The composites’ catalytic performance is evaluated through separate acid and base reactions, followed by a combined cascade of a deacetalization−Knoevenagel condensation for the synthesis of chalcone compounds. High initial reaction rates are demonstrated, but modest overall cascade product formation rates are observed, attributed to interactions between Brønsted acid sites and base amine groups that occur in the polymer-grafted systems. Physical mixtures of NaH-ZNT-SH and poly(oxazoline)s, lacking covalent linkages between ZNT and the polymer, support this supposition. This work demonstrates the potential of NaH-ZNT-poly(oxazoline) composites for liquid-phase cascade catalysis for synthesizing compounds of potential medicinal interest, highlighting the benefits of the grafting-to approach as well as the need for further optimization of the catalytic performance.

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

In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5’-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.

36 MATERIALS SCIENCE↗
DOE OSTI2026

Local Structural Coherence and Interfacial Charge Transfer in Cu 2 ⁢S/Mo⁢S 2 Heterostructure

Precise control over electronic coupling at nanoscale interfaces is critical for designing materials with tunable charge-transfer behavior and catalytic function. Heterostructures with locally coherent interfaces provide a platform for interrogating interfacial charge redistribution in coupled material systems. Here, we report Cu 2 ⁢S/Mo⁢S 2 heterostructures exhibiting nanoscale crystallographic alignment, which are synthesized through a rapid thermal transformation pathway. We employed electrochemical reduction reactions to probe interfacial charge transfer, revealing shifts in product distribution attributable to modified interfacial energetics, even in the absence of optimized catalytic performance. The observed formate Faradaic efficiency suggests that interfacial electronic modulation in the heterostructure shifts product selectivity towards formate, highlighting how interface-driven electronic modulation can direct reaction pathways and influence product selectivity. Optimizing catalyst loading, architecture, and reactor configuration will be critical for future improvement. Structural and compositional integrity were confirmed through powder x-ray diffraction, x-ray photoelectron spectroscopy, and high-resolution transmission electron microscopy. Electron transfer between Cu 2 ⁢S and Mo⁢S 2 domains was further evaluated by electrochemical impedance spectroscopy, while selected-area electron diffraction revealed local crystallographic alignment consistent with a local epitaxial relationship at the Cu 2 S/Mo⁢S 2 heterointerface. To illustrate the broader applicability of this approach, a Zn⁢S/Mo⁢S 2 heterostructure was also synthesized using the same microwave strategy, confirming the generalizability of interfacial engineering principles across metal sulfide-Mo⁢S 2 systems. Collectively, these findings demonstrate that controlled local epitaxial alignment serves as an effective design principle for tuning interfacial energetics and catalytic reactivity in complex heterostructure materials.

carbon capture & utilization↗
DOE OSTI2023

Mycobacterium tuberculosis CitA activity is modulated by cysteine oxidation and pyruvate binding

As an adaptation for survival during infection, Mycobacterium tuberculosis becomes dormant, reducing its metabolism and growth. Two types of citrate synthases have been identified in Mycobacterium tuberculosis, GltA2 and CitA. Previous work shows that overexpression of CitA, the secondary citrate synthase, stimulates the growth of Mycobacterium tuberculosis under hypoxic conditions without showing accumulation of triacylglycerols and makes mycobacteria more sensitive to antibiotics, suggesting that CitA may play a role as a metabolic switch during infection and may be an interesting TB drug target. To assess the druggability and possible mechanisms of targeting CitA with small-molecule compounds, the CitA crystal structure was solved to 2.1 Å by X-ray crystallography. The solved structure shows that CitA lacks an NADH binding site that would afford allosteric regulation, which is atypical of most citrate synthases. However, a pyruvate molecule is observed within the analogous domain, suggesting pyruvate may instead be the allosteric regulator for CitA. Further, the R149 and R153 residues forming the charged portion of the pyruvate binding pocket were mutated to glutamate and methionine, respectively, to assess the effect of mutations on activity. Protein thermal shift assay shows thermal stabilization of CitA in the presence of pyruvate compared to the two CitA variants designed to decrease pyruvate affinity. Solved crystal structures of both variants show no significant structural changes. However, the catalytic efficiency of the R153M variant increases by 2.6-fold. Additionally, we show that covalent modification of C143 of CitA by Ebselen completely arrests enzyme activity. Similar inhibition is observed using two spirocyclic Michael acceptor containing compounds, which inhibit CitA with IC app 50 values of 6.6 and 10.9 μM. A crystal structure of CitA modified by Ebselen was solved, but significant structural changes were lacking. Considering that covalent modification of C143 inactivates CitA and the proximity of C143 to the pyruvate binding site, this suggests that structural and/or chemical changes in this sub-domain are responsible for regulating CitA enzymatic activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Exploring the mobility of Cu in bimetallic nanocrystals to promote atomic-scale transformations under a reactive gas environment

Bimetallic nanocrystals (NCs) often show improved catalytic activities compared to their monometallic counterparts, but to optimize the performance it is crucial to understand how they behave under actual reaction conditions, i.e. in gas environments. Here, in this study, we use powder X-ray diffraction (PXRD), total scattering (TS) with pair distribution function (PDF) analysis and in situ high-resolution transmission electron microscopy (HR-TEM) to provide new insights into the atomic-scale behaviour of NC catalysts under a reactive gas environment. By investigating Au, Cu, Pd, PdCu, AuPd and AuCu NCs, we observe that the properties of bimetallic NCs differ significantly from their monometallic counterparts. While metal oxide phases formed for monometallic Pd and Cu under O 2 -exposure, bimetallic PdCu and AuCu NCs showed loss of metallic Cu in the crystalline phases after exposure to O 2 . However, upon introducing the bimetallic NCs to a reducing atmosphere, the Cu was found to reappear and reincorporate into a crystalline phase, forming the initial bimetallic structures. By combining TS, PDF analysis and in situ HR-TEM, we saw that Cu segregates to the NC surfaces or forms small CuO domains under O 2 -exposure. Our results thus indicate that the Cu mobility promotes segregation and formation of CuO along with the formation of a monometallic phase, which ultimately changes the resulting active surface sites of the nanocatalyst. Understanding the dynamical structure–property relations of nanocatalysts is key to enable rational design of efficient and robust catalysts for controlled catalytic reactions.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Evolutionary Diversification of Host-Targeted Bartonella Effectors Proteins Derived from a Conserved FicTA Toxin-Antitoxin Module

Proteins containing a FIC domain catalyze AMPylation and other post-translational modifications (PTMs). In bacteria, they are typically part of FicTA toxin-antitoxin modules that control conserved biochemical processes such as topoisomerase activity, but they have also repeatedly diversified into host-targeted virulence factors. Among these, Bartonella effector proteins (Beps) comprise a particularly diverse ensemble of FIC domains that subvert various host cellular functions. However, no comprehensive comparative analysis has been performed to infer molecular mechanisms underlying the biochemical and functional diversification of FIC domains in the vast Bep family. Here, we used X-ray crystallography, structural modelling, and phylogenetic analyses to unravel the expansion and diversification of Bep repertoires that evolved in parallel in three Bartonella lineages from a single ancestral FicTA toxin-antitoxin module. Our analysis is based on 99 non-redundant Bep sequences and nine crystal structures. Inferred from the conservation of the FIC signature motif that comprises the catalytic histidine and residues involved in substrate binding, about half of them represent AMP transferases. A quarter of Beps show a glutamate in a strategic position in the putative substrate binding pocket that would interfere with triphosphate-nucleotide binding but may allow binding of an AMPylated target for deAMPylation or another substrate to catalyze a distinct PTM. The β-hairpin flap that registers the modifiable target segment to the active site exhibits remarkable structural variability. The corresponding sequences form few well-defined groups that may recognize distinct target proteins. The binding of Beps to promiscuous FicA antitoxins is well conserved, indicating a role of the antitoxin to inhibit enzymatic activity or to serve as a chaperone for the FIC domain before translocation of the Bep into host cells. Taken together, our analysis indicates a remarkable functional plasticity of Beps that is mostly brought about by structural changes in the substrate pocket and the target dock. These findings may guide future structure–function analyses of the highly versatile FIC domains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Valorizing the carbon byproduct of methane pyrolysis in batteries

While low-cost natural gas remains abundant, the energy content of this fuel can be utilized without greenhouse gas emissions through the production of molecular hydrogen and solid carbon via methane pyrolysis. In the absence of a carbon tax, methane pyrolysis is not economically competitive with current hydrogen production methods unless the carbon byproducts can be valorized. In this work, we assess the viability of the carbon byproduct produced from methane pyrolysis in molten salts as high-value-added anode or conductive additive for secondary Li-ion and Na-ion batteries. Raman characterization and electrochemical differential capacity analysis demonstrate that the use of molten salt mixtures with catalytically-active FeCl 3 - or MnCl 2 result in more graphitic carbon co-products. These graphitic carbons exhibit the best electrochemical performance (up to 272 mAh/g of reversible capacity) when used as Li-ion anodes. For all carbon samples studied here, disordered carbon domains and retained salt species trapped and/or intercalated into the carbon structure were identified by X-ray photoelectron and multinuclear solid-state nuclear magnetic resonance spectroscopy. The latter lead to reduced electrochemical activity and reversibility, and poorer rate performance compared to commercial carbon anodes. The electronic conductivity of the pyrolyzed carbons is found to be highly dependent on their purity, with the purest carbon exhibiting an electronic conductivity nearly on par with that of commercial carbon additives. These findings suggest that more effective removal of the salt catalyst could enable applications of these carbons in secondary batteries, providing a financial incentive for the large-scale implementation of methane pyrolysis for “low-carbon” hydrogen production.

08 HYDROGEN↗
DOE OSTI2021

Structure of Pseudomonas aeruginosa spermidine dehydrogenase: a polyamine oxidase with a novel heme‐binding fold

The opportunistic pathogen Pseudomonas aeruginosa can utilize polyamines (including putrescine, cadaverine, 4‐aminobutyrate, spermidine, and spermine) as its sole source of carbon and nitrogen. Spermidine dehydrogenase (SpdH) is a component of one of the two polyamine utilization pathways identified in P . aeruginosa , but little is known about its structure and function. Here, we report the first crystal structure of SpdH from P . aeruginosa to 1.85 Å resolution. The resulting core structure confirms that SpdH belongs to the polyamine oxidase (PAO) family with flavin‐binding and substrate‐binding domains. A unique N‐terminal extension wraps around the flavin‐binding domain of SpdH and is required for heme binding, placing a heme cofactor in close proximity to the FAD cofactor. Structural and mutational analysis reveals that residues in the putative active site at the re side of the FAD isoalloxazine ring form part of the catalytic machinery. PaSpdH features an unusual active site and lacks the conserved lysine that forms part of a lysine–water–flavin N5 atom interaction in other PAO enzymes characterized to date. Mutational analysis further confirms that heme is required for catalytic activity. This work provides an important starting point for understanding the role of SpdH, which occurs universally in P . aeruginosa strains, in polyamine metabolism.

Che, Shiyou↗
DOE OSTI2020

Structural basis of peptidoglycan endopeptidase regulation

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae. Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Maturation of Rhodobacter capsulatus Multicopper Oxidase CutO Depends on the CopA Copper Efflux Pathway and Requires the cutF Product

Copper (Cu) is an essential cofactor required for redox enzymes in all domains of life. Because of its toxicity, tightly controlled mechanisms ensure Cu delivery for cuproenzyme biogenesis and simultaneously protect cells against toxic Cu. Many Gram-negative bacteria contain extracytoplasmic multicopper oxidases (MCOs), which are involved in periplasmic Cu detoxification. MCOs are unique cuproenzymes because their catalytic center contains multiple Cu atoms, which are required for the oxidation of Cu 1+ to the less toxic Cu 2+ . Hence, Cu is both substrate and essential cofactor of MCOs. Here, we investigated the maturation of Rhodobacter capsulatus MCO CutO and its role in periplasmic Cu detoxification. A survey of CutO activity of R. capsulatus mutants with known defects in Cu homeostasis and in the maturation of the cuproprotein cbb 3 -type cytochrome oxidase ( cbb 3 -Cox) was performed. This revealed that CutO activity is largely independent of the Cu-delivery pathway for cbb 3 -Cox biogenesis, except for the cupric reductase CcoG, which is required for full CutO activity. The most pronounced decrease of CutO activity was observed with strains lacking the cytoplasmic Cu chaperone CopZ, or the Cu-exporting ATPase CopA, indicating that CutO maturation is linked to the CopZ-CopA mediated Cu-detoxification pathway. Our data demonstrate that CutO is important for cellular Cu resistance under both aerobic and anaerobic growth conditions. CutO is encoded in the cutFOG operon, but only CutF, and not CutG, is essential for CutO activity. No CutO activity is detectable when cutF or its putative Cu-binding motif are mutated, suggesting that the cutF product serves as a Cu-binding component required for active CutO production. Bioinformatic analyses of CutF-like proteins support their widespread roles as putative Cu-binding proteins for several Cu-relay pathways. Our overall findings show that the cytoplasmic CopZ-CopA dependent Cu detoxification pathway contributes to providing Cu to CutO maturation, a process that strictly relies on cutF .

59 BASIC BIOLOGICAL SCIENCES↗