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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 1,729 records · Page 96
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Engineering and characterization of carbohydrate‐binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts

Abstract Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM‐polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM‐based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type‐A CBMs from families 3a and 64 with crystalline cellulose‐I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose‐I using equilibrium binding assays. To compute the adsorption ( nk on ) and desorption ( k off ) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide‐field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.

09 BIOMASS FUELS↗
DOE OSTI2019

Peatland microbial community response to altered climate tempered by nutrient availability

Northern latitude peatlands contain large reserves of soil carbon (C) due in part to climatic constraints on decomposition, including low temperatures and water inundated soils. Under future climate change scenarios these peatlands will experience warmer temperatures, extended growing seasons, and a potential draw down of the water table, all of which improve conditions for decomposition, as well as photosynthesis. Increased photosynthesis may feedback to increase decomposition of soil C through increased root exudates to belowground decomposer communities, primarily as low molecular weight carbon compounds (LMWCC). In this study, we examine the interactive effects of climate, a combination of three temperatures and two moisture regimes, and root exudates on microbial decomposer function, measured as CO2 respiration, biomass, and potential enzyme activity. We had four substrate treatments: two common LMWCC (glycine or glucose + citric acid), chitin to simulate fungal necromass, and DI as the control. Our results support our first hypothesis that increasing temperature will increase C respiration across substrate and moisture treatments. Our second hypothesis was that compared to control and chitin, soluble substrates (i.e., LMWCC) will enhance respiration across all climate treatments. This was only partially supported. As expected, the two LMWCC substrate additions increased C respiration above the two other substrate additions at current recorded growing season average (12 °C, low treatment) and high (20 °C, med treatment) temperature treatments. Surprisingly, when the system was pushed to a higher temperature extreme (28 °C, high treatment), the low moisture controls respired more C than the other substrate × climate treatments. Potential enzyme activity and demand for phosphorus appear to explain these trends as opposed to changes to microbial biomass. Our results indicate that under projected future high temperatures, the peatland microbial community allocates additional labile C resources to enzyme production to meet nutrient demands, and as such, dampens C lost through respiration.

Keiser, Ashley D.↗
DOE OSTI2015

Structure of a eukaryotic SWEET transporter in a homotrimeric complex

Eukaryotes rely on efficient distribution of energy and carbon skeletons between organs in the form of sugars. Glucose in animals and sucrose in plants serve as the dominant distribution forms. Cellular sugar uptake and release require vesicular and/or plasma membrane transport proteins. Humans and plants use proteins from three superfamilies for sugar translocation: the major facilitator superfamily (MFS), the sodium solute symporter family (SSF; only in the animal kingdom), and SWEETs. SWEETs carry mono- and disaccharides across vacuolar or plasma membranes. Plant SWEETs play key roles in sugar translocation between compartments, cells, and organs, notably in nectar secretion, phloem loading for long distance translocation, pollen nutrition, and seed filling. Plant SWEETs cause pathogen susceptibility possibly by sugar leakage from infected cells. The vacuolar Arabidopsis thaliana AtSWEET2 sequesters sugars in root vacuoles; loss-of-function mutants show increased susceptibility to Pythium infection. In this paper, we show that its orthologue, the vacuolar glucose transporter OsSWEET2b from rice (Oryza sativa), consists of an asymmetrical pair of triple-helix bundles, connected by an inversion linker transmembrane helix (TM4) to create the translocation pathway. Structural and biochemical analyses show OsSWEET2b in an apparent inward (cytosolic) open state forming homomeric trimers. TM4 tightly interacts with the first triple-helix bundle within a protomer and mediates key contacts among protomers. Structure-guided mutagenesis of the close paralogue SWEET1 from Arabidopsis identified key residues in substrate translocation and protomer crosstalk. Finally, insights into the structure–function relationship of SWEETs are valuable for understanding the transport mechanism of eukaryotic SWEETs and may be useful for engineering sugar flux.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Structural insights into inhibition of lipid I production in bacterial cell wall synthesis

Antibiotic-resistant bacterial infection is a serious threat to public health. Peptidoglycan biosynthesis is a well-established target for antibiotic development. MraY (phospho-MurNAc-pentapeptide translocase) catalyses the first and an essential membrane step of peptidoglycan biosynthesis. It is considered a very promising target for the development of new antibiotics, as many naturally occurring nucleoside inhibitors with antibacterial activity target this enzyme. However, antibiotics targeting MraY have not been developed for clinical use, mainly owing to a lack of structural insight into inhibition of this enzyme. Here we present the crystal structure of MraY from Aquifex aeolicus (MraY AA ) in complex with its naturally occurring inhibitor, muraymycin D2 (MD2). We show that after binding MD2, MraY AA undergoes remarkably large conformational rearrangements near the active site, which lead to the formation of a nucleoside-binding pocket and a peptide-binding site. MD2 binds the nucleoside-binding pocket like a two-pronged plug inserting into a socket. Further interactions it makes in the adjacent peptide-binding site anchor MD2 to and enhance its affinity for MraY AA . Surprisingly, MD2 does not interact with three acidic residues or the Mg 2+ cofactor required for catalysis, suggesting that MD2 binds to MraY AA in a manner that overlaps with, but is distinct from, its natural substrate, UDP-MurNAc-pentapeptide. We have determined the principles of MD2 binding to MraY AA , including how it avoids the need for pyrophosphate and sugar moieties, which are essential features for substrate binding. The conformational plasticity of MraY could be the reason that it is the target of many structurally distinct inhibitors. Furthermore, these findings can inform the design of new inhibitors targeting MraY as well as its paralogues, WecA and TarO.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Jungle Express is a versatile repressor system for tight transcriptional control

Abstract Tightly regulated promoters are essential for numerous biological applications, where strong inducibility, portability, and scalability are desirable. Current systems are often incompatible with large-scale fermentations due to high inducer costs and strict media requirements. Here, we describe the bottom-up engineering of ‘Jungle Express’, an expression system that enables efficient gene regulation in diverse proteobacteria. This system is guided by EilR, a multidrug-binding repressor with high affinity to its optimized operator and cationic dyes that act as powerful inducers at negligible costs. In E. coli , the engineered promoters exhibit minimal basal transcription and are inducible over four orders of magnitude by 1 µM crystal violet, reaching expression levels exceeding those of the strongest current bacterial systems. Further, we provide molecular insights into specific interactions of EilR with its operator and with two inducers. The versatility of Jungle Express opens the way for tightly controlled and efficient gene expression that is not restricted to host organism, substrate, or scale.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Interaction of surface and bulk acoustic waves with a two-dimensional semimetal

The interaction of a surface elastic Rayleigh wave with an electron-hole plasma in a two-dimensional semimetal has been theoretically studied as determined by the deformation potential and piezoelectric mechanisms. Dispersion equations describing the coupled plasmon-acoustic modes for both types of interaction are derived, and damping of the Rayleigh wave is calculated. The damping of the acoustic and optical plasmon modes, which is related to the sound emission by plasma oscillations into the substrate volume, is calculated and it is shown that this sound emission is predominantly determined by the acoustic plasmon mode in the case of a deformation potential mechanism and by the optical mode in the case of a piezoelectric mechanism.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
NASA NTRS2012

On-chip polarimetry for high-throughput screening of nanoliter and smaller sample volumes

A polarimetry technique for measuring optical activity that is particularly suited for high throughput screening employs a chip or substrate (22) having one or more microfluidic channels (26) formed therein. A polarized laser beam (14) is directed onto optically active samples that are disposed in the channels. The incident laser beam interacts with the optically active molecules in the sample, which slightly alter the polarization of the laser beam as it passes multiple times through the sample. Interference fringe patterns (28) are generated by the interaction of the laser beam with the sample and the channel walls. A photodetector (34) is positioned to receive the interference fringe patterns and generate an output signal that is input to a computer or other analyzer (38) for analyzing the signal and determining the rotation of plane polarized light by optically active material in the channel from polarization rotation calculations.

Bornhop, Darryl J.↗
DOE OSTI2014

Effect of superconducting spacer layer thickness on magneto-transport and magnetic properties of La{sub 0.7}Sr{sub 0.3}MnO{sub 3}/YBa{sub 2}Cu{sub 3}O{sub 7}/La{sub 0.7}Sr{sub 0.3}MnO{sub 3} heterostructures

We have studied the magneto-transport and magnetic properties of LSMO/YBCO/LSMO trilayers on LaAlO{sub 3} (001) substrate, deposited using pulsed laser deposition technique. From x-ray diffraction measurements, it is confirmed that the grown trilayer films are single phase natured. The temperature dependent resistivity shows a metallic behavior below 350 K. At low temperature from resistivity fitted data, we observe that electron-electron, electron-phonon, and electron-magnon interactions are the main factors for scattering of carriers. The ferromagnetic LSMO layers suppress the critical temperature of YBCO spacer layer. We observe maximum magnetoresistance value ∼49% at 250 K for LSMO(200 nm)/YBCO(50 nm)/LSMO(200 nm) trilayer. Magnetization measurements reveal that at room temperature the YBCO spacer layer is allowing the LSMO layers to interact antiferromagnetically.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2026

Selective Conversion of CO 2 to Methanol on a In 2 O 3– x –TiO 2 (110) Interface: Importance of Oxide–Oxide Interactions

Methanol is a strategic energy vector for the storage and delivery of energy and is a widely used precursor for the synthesis of many high-value chemicals. The hydrogenation of carbon dioxide (CO 2 ) into methanol is a key process in industrial operations. Here, in this study, we show that an oxide-oxide interface generated by a low loading (0.15 ML) of In 2 O 3-x on a TiO 2 (110) substrate has a high activity and selectivity as a catalyst for the CO 2 + 3H 2 → CH 3 OH + H 2 O process. The properties of the In 2 O 3-x -TiO 2 interface under reaction conditions were investigated using a combination of synchrotron-based ambient pressure X-ray photoelectron spectroscopy (AP-XPS), temperature programmed desorption (TPD), and catalytic testing. The In 2 O 3-x overlayer spread out on top of the titania and was rich in defects and O vacancies that activated CO 2 and H 2 as reactants, without destroying CH 3 O and CH 3 OH as reaction products. The In 2 O 3-x /TiO 2 (110) catalyst is at least one order of magnitude more active than bulk indium oxide while maintaining a very high selectivity (~80%) towards methanol production. Under the rich hydrogen environment of methanol synthesis, the oxide-oxide interactions allowed only a partial reduction of the In cations, preventing the formation of metal alloys as seen in the case of catalysts with metal-indium oxide interfaces. Thus, the dispersion of low loadings of In 2 O 3-x on a stable oxide substrate is a valid and low-cost approach for generating efficient catalysts for CO 2 valorization.

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

Formation of a carbon nanofilm on oil-coated KU-1 glass annealed by KrF laser radiation

The annealing of an oil layer on the output face of a KU-1 glass substrate in air by a high-power 80 ns KrF laser pulse is studied. A polymer-like hydrocarbon film up to 40 nm thick is found to form at laser radiation fluences F above 2 J cm{sup −2} on the glass surface. The upper limit of F ≈ 6 J cm{sup −2} is determined by the damage of the glass samples whose size is approximately equal to the laser spot diameter (1 cm). (interaction of laser radiation with matter. laser plasma)

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2016

Magnetic and optical properties of Mn-doped ZnO vertically aligned nanorods synthesized by hydrothermal technique

In this paper we have reported the synthesis of high quality vertically aligned undoped and Mn-doped ZnO single crystalline nanorods arrays on Si (100) substrates using two steps process, namely, initial slow seed layer formation followed by solution growth employing wet chemical hydrothermal method. The shapes of the as grown single crystalline nanorods are hexagonal. The diameter and length of the as grown undoped ZnO nanorods varies in the range of 80-150 nm and 1.0 - 1.4 μm, respectively. Along with the lattice parameters of the hexagonal crystal structure, the diameter and length of Mn doped ZnO nanorods are found to increase slightly as compared to the undoped ZnO nanorods. The X-ray photoelectron spectroscopy confirms the presence of Mn atoms in Mn{sup 2+} state in the single crystalline ZnO nanorods. The recorded photoluminescence spectrum contains two emissions peaks having UV exciton emissions along with a green-yellow emission. The green-yellow emissions provide the evidence of singly ionized oxygen vacancies. The magnetic field dependent magnetization measurements [M (H)] and zero field cooled (ZFC) and field cooled (FC) magnetization [M(T)] measurements have been carried out at different isothermal conditions in the temperature range of 5-300 K. The Mn doped ZnO nanorods clearly show room temperature ferromagnetic ordering near room temperature down to 5 K. The observed magnetization may be attributed to the long range ferromagnetic interaction between bound magnetic polarons led by singly charged oxygen vacancies.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2013

The role of the (111) texture on the exchange bias and interlayer coupling effects observed in sputtered NiFe/IrMn/Co trilayers

Magnetic properties of sputtered NiFe/IrMn/Co trilayers grown on different seed layers (Cu or Ta) deposited on Si (100) substrates were investigated by magnetometry and ferromagnetic resonance measurements. Exchange bias effect and magnetic spring behavior have been studied by changing the IrMn thickness. As shown by X-ray diffraction, Ta and Cu seed layers provoke different degrees of (111) fcc-texture that directly affect the exchange bias and indirectly modify the exchange spring coupling behavior. Increasing the IrMn thickness, it was observed that the coupling angle between the Co and NiFe ferromagnetic layers increases for the Cu seed system, but it reduces for the Ta case. The results were explained considering (i) different anisotropies of the Co and IrMn layers induced by the different degree of the (111) texture and (ii) the distinct exchange bias set at the NiFe/IrMn and IrMn/Co interfaces in both systems. The NiFe and Co interlayer coupling angle is strongly correlated with both exchange bias and exchange magnetic spring phenomena. It was also shown that the highest exchange bias field occurs when an unstressed L1{sub 2} IrMn structure is stabilized.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2017

Laser-induced modification of graphene in the presence of ethanol on a graphene – substrate interface

We have studied the effect that the substitution of an organic substance (ethanol) for water adsorbate on a CVD graphene–SiO{sub 2}/Si interface has on the laser-induced modification of graphene and graphene structures on the SiO{sub 2} film. Scanning probe microscopy has been used to analyse changes in the electronic properties of graphene structures on a hydrophilic substrate in the presence of ethanol and as a result of a laser-induced spatial redistribution of a water–alcohol adsorbate on the interface. It has been demonstrated experimentally that ethanol substitution for water adsorbate leads to an increase in the surface potential of the graphene, which is equivalent to a reduction in its work function with respect to the original level under normal conditions at a relative humidity of air from 30% to 60%. In the laser irradiation zone, we observe an additional increase in surface potential by 30–50 mV. Thus, ethanol makes it possible to tune the laser-induced electronic properties of graphene on a substrate. In addition, it has been shown that the intercalation of ethanol molecules leads to severe temporal instability of the physical properties of graphene structures produced by local laser irradiation. We have demonstrated the possibility of information ‘rewriting’ by low-intensity laser pulses in microregions with a changed surface potential in the presence of ethanol. (interaction of laser radiation with matter. laser plasma)

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2012

Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors

Clinical inhibitor amprenavir (APV) is less effective on HIV-2 protease (PR{sub 2}) than on HIV-1 protease (PR{sub 1}). We solved the crystal structure of PR{sub 2} with APV at 1.5 {angstrom} resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR{sub 1} mutant (PR{sub 1M}) with substitutions V32I, I47V, and V82I that mimic the inhibitor binding site of PR{sub 2}. PR{sub 1M} more closely resembled PR{sub 2} than PR{sub 1} in catalytic efficiency on four substrate peptides and inhibition by APV, whereas few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR{sub 1M} with APV, DRV, and SQV were compared with available PR{sub 1} and PR{sub 2} complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR{sub 1M} and PR{sub 1}, however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR{sub 1M}. Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, whereas Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR{sub 1M} and PR{sub 2} relative to the strong hydrogen bonds observed in PR{sub 1}, consistent with 15- and 19-fold weaker inhibition, respectively. Overall, PR{sub 1M} partially replicates the specificity of PR{sub 2} and gives insight into drug resistant mutations at residues 32, 47, and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV-2.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Aligned and exchange-coupled L1{sub 0} (Fe,Co)Pt-based magnetic films

Films of aligned L1{sub 0}-structure (Fe,Co)Pt with fcc Fe(Co,Pt) are synthesized by co-sputtering Fe, Co, and Pt on an (001) MgO substrate with in situ heating at 830 deg. C. The nanostructures and magnetic properties of the films are characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID). The compositions of the samples (Fe,Co){sub x}Pt{sub 1-x} are designed to maintain an atomic Fe: Co ratio of 65: 35 while increasing the Fe,Co content in each successive sample. In samples with low Fe and Co concentration, the XRD patterns exhibit three strong peaks, namely L1{sub 0} (Fe,Co)Pt (001), L1{sub 0} (Fe,Co)Pt (002), and MgO (002). A fourth peak is observed in samples with high Fe and Co concentration and identified as fcc (002). The XRD patterns confirm the formation of L1{sub 0}-ordered (Fe,Co)Pt and its epitaxial growth on MgO. TEM shows that the (Fe,Co)Pt films form isolated magnetic grains of about 100 nm in diameter. Hysteresis-loop measurements show that the increase of the Fe,Co concentration from 57.3 to 68.3 at % enhances the saturation magnetization M{sub s} from 1245 emu/cm{sup 3} to 1416 emu/cm{sup 3}, and the coercivity decreases from 32 kOe to 8.9 kOe. The nominal maximum energy product per grain is 64 MGOe.

36 MATERIALS SCIENCE↗
DOE OSTI2014

Molecular Electronic Level Alignment at Weakly Coupled Organic Film/Metal Interfaces

Electronic level alignment at interfaces of molecular materials with inorganic semiconductors and metals controls many interfacial phenomena. How the intrinsic properties of the interacting systems define the electronic structure of their interface remains one of the most important problems in molecular electronics and nanotechnology that can be solved through a combination of surface science experimental techniques and theoretical modeling. In this article, we address this fundamental problem through experimental and computational studies of molecular electronic level alignment of thin films of C6F6 on noble metal surfaces. The unoccupied electronic structure of C6F6 is characterized with single molecule resolution using low-temperature scanning tunneling microscopy-based constant-current distance-voltage spectroscopy. The experiments are performed on several noble metal surfaces with different work functions and distinct surface-normal projected band structures. In parallel, the electronic structures of the quantum wells (QWs) formed by the lowest unoccupied molecular orbital state of the C6F6 monolayer and multilayer films and their alignment with respect to the vacuum level of the metallic substrates are calculated by solving the Schrödinger equation for a semiempirical one-dimensional (1D) potential of the combined system using input from density functional theory. Our analysis shows that the level alignment for C6F6 molecules bound through weak van der Waals interactions to noble metal surfaces is primarily defined by the image potential of metal, the electron affinity of the molecule, and the molecule surface distance. We expect the same factors to determine the interfacial electronic structure for a broad range of molecule/metal interfaces.

Environmental Molecular Sciences Laboratory↗
DOE OSTI2013

Inhibition of CYP1 by berberine, palmatine, and jatrorrhizine: Selectivity, kinetic characterization, and molecular modeling

Cytochrome P450 (P450, CYP) 1 family plays a primary role in the detoxification and bioactivation of polycyclic aromatic hydrocarbons. Human CYP1A1, CYP1A2, and CYP1B1 exhibit differential substrate specificity and tissue distribution. Berberine, palmatine, and jatrorrhizine are protoberberine alkaloids present in several medicinal herbs, such as Coptis chinensis (Huang-Lian) and goldenseal. These protoberberines inhibited CYP1A1.1- and CYP1B1.1-catalyzed 7-ethoxyresorufin O-deethylation (EROD) activities, whereas CYP1A2.1 activity was barely affected. Kinetic analysis revealed that berberine noncompetitively inhibited EROD activities of CYP1A1.1 and CYP1B1.1, whereas palmatine and jatrorrhizine caused either competitive or mixed type of inhibition. Among protoberberines, berberine caused the most potent and selective inhibitory effect on CYP1B1.1 with the least K{sub i} value of 44 ± 16 nM. Berberine also potently inhibited CYP1B1.1 activities toward 7-ethoxycoumarin and 7-methoxyresorufin, whereas the inhibition of benzo(a)pyrene hydroxylation activity was less pronounced. Berberine inhibited the polymorphic variants, CYP1B1.3 (V432L) and CYP1B1.4 (N453S), with IC{sub 50} values comparable to that for CYP1B1.1 inhibition. Berberine-mediated inhibition was abolished by a mutation of Asn228 to Thr in CYP1B1.1, whereas the inhibition was enhanced by a reversal mutation of Thr223 to Asn in CYP1A2.1. This result in conjugation with the molecular modeling revealed the crucial role of hydrogen-bonding interaction of Asn228 on CYP1B1.1 with the methoxy moiety of berberine. These findings demonstrate that berberine causes a selective CYP1B1-inhibition, in which Asn228 appears to be crucial. The inhibitory effects of berberine on CYP1B1 activities toward structurally diverse substrates can be different. - Highlights: • Berberine preferentially inhibited CYP1B1 activity. • Berberine caused similar inhibitory effects on CYP1B1.1, CYP1B1.3 and CYP1B1.4. • Asn228 in CYP1B1 was an important residue for berberine-mediated inhibition.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Mechanism of N[superscript 10]-formyltetrahydrofolate synthetase derived from complexes with intermediates and inhibitors

N{sup 10}-formyltetrahydrofolate synthetase (FTHFS) is a folate enzyme that catalyzes the formylation of tetrahydrofolate (THF) in an ATP dependent manner. Structures of FTHFS from the thermophilic homoacetogen, Moorella thermoacetica, complexed with (1) a catalytic intermediate-formylphosphate (XPO) and product-ADP; (2) with an inhibitory substrate analog-folate; (3) with XPO and an inhibitory THF analog, ZD9331, were used to analyze the enzyme mechanism. Nucleophilic attack of the formate ion on the gamma phosphate of ATP leads to the formation of XPO and the first product ADP. A channel that leads to the putative formate binding pocket allows for the binding of ATP and formate in random order. Formate binding is due to interactions with the gamma-phosphate moiety of ATP and additionally to two hydrogen bonds from the backbone nitrogen of Ala276 and the side chain of Arg97. Upon ADP dissociation, XPO reorients and moves to the position previously occupied by the beta-phosphate of ATP. Conformational changes that occur due to the XPO presence apparently allow for the recruitment of the third substrate, THF, with its pterin moiety positioned between Phe384 and Trp412. This position overlaps with that of the bound nucleoside, which is consistent with a catalytic mechanism hypothesis that FTHFS works via a sequential ping-pong mechanism. More specifically, a random bi uni uni bi ping-pong ter ter mechanism is proposed. Additionally, the native structure originally reported at a 2.5 {angstrom} resolution was redetermined at a 2.2 {angstrom} resolution.

08 HYDROGEN↗