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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 145 records · Page 8
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Structure and mechanism of Staphylococcus aureus oleate hydratase (OhyA)

Flavin adenine dinucleotide (FAD)-dependent bacterial oleate hydratases (OhyAs) catalyze the addition of water to isolated fatty acid carbon–carbon double bonds. Staphylococcus aureus uses OhyA to counteract the host innate immune response by inactivating antimicrobial unsaturated fatty acids. Mechanistic information explaining how OhyAs catalyze regiospecific and stereospecific hydration is required to understand their biological functions and the potential for engineering new products. In this study, we deduced the catalytic mechanism of OhyA from multiple structures of S. aureus OhyA in binary and ternary complexes with combinations of ligands along with biochemical analyses of relevant mutants. The substrate-free state shows Arg81 is the gatekeeper that controls fatty acid entrance to the active site. FAD binding engages the catalytic loop to simultaneously rotate Glu82 into its active conformation and Arg81 out of the hydrophobic substrate tunnel, allowing the fatty acid to rotate into the active site. FAD binding also dehydrates the active site, leaving a single water molecule connected to Glu82. This active site water is a hydronium ion based on the analysis of its hydrogen bond network in the OhyA•PEG400•FAD complex. We conclude that OhyA accelerates acid-catalyzed alkene hydration by positioning the fatty acid double bond to attack the active site hydronium ion, followed by the addition of water to the transient carbocation intermediate. Structural transitions within S. aureus OhyA channel oleate to the active site, curl oleate around the substrate water, and stabilize the hydroxylated product to inactivate antimicrobial fatty acids.

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

Comparative proteomic analysis of Desulfotomaculum reducens MI-1: Insights into the metabolic versatility of a gram-positive sulfate- and metal-reducing bacterium

In this study, the proteomes of the metabolically versatile and poorly characterized Gram-positive bacterium Desulfotomaculum reducens MI-1 were compared across four cultivation conditions including sulfate reduction, soluble Fe(III) reduction, insoluble Fe(III) reduction, and pyruvate fermentation. Collectively across conditions, we observed at high confidence ~38% of genome-encoded proteins. Here, we focus on proteins that display significant differential abundance on conditions tested. To the best of our knowledge, this is the first full-proteome study focused on a Gram-positive organism cultivated either on sulfate or metal-reducing conditions. Several proteins with uncharacterized function encoded within heterodisulfide reductase ( hdr )-containing loci were upregulated on either sulfate (Dred_0633-4, Dred_0689-90, and Dred_1325-30) or Fe(III)-citrate-reducing conditions (Dred_0432-3 and Dred_1778-84). Two of these hdr -containing loci display homology to recently described flavin-based electron bifurcation (FBEB) pathways (Dred_1325-30 and Dred_1778-84). Additionally, we propose that a cluster of proteins, which is homologous to a described FBEB lactate dehydrogenase (LDH) complex, is performing lactate oxidation in D. reducens (Dred_0367-9). Analysis of the putative sulfate reduction machinery in D. reducens revealed that most of these proteins are constitutively expressed across cultivation conditions tested. In addition, peptides from the single multiheme c-type cytochrome (MHC) in the genome were exclusively observed on the insoluble Fe(III) condition, suggesting that this MHC may play a role in reduction of insoluble metals.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1992

Isolation, characterization, and amino acid sequences of auracyanins, blue copper proteins from the green photosynthetic bacterium Chloroflexus aurantiacus

Three small blue copper proteins designated auracyanin A, auracyanin B-1, and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus. All three auracyanins are peripheral membrane proteins. Auracyanin A was described previously (Trost, J. T., McManus, J. D., Freeman, J. C., Ramakrishna, B. L., and Blankenship, R. E. (1988) Biochemistry 27, 7858-7863) and is not glycosylated. The two B forms are glycoproteins and have almost identical properties to each other, but are distinct from the A form. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis apparent monomer molecular masses are 14 (A), 18 (B-2), and 22 (B-1) kDa. The amino acid sequences of the B forms are presented. All three proteins have similar absorbance, circular dichroism, and resonance Raman spectra, but the electron spin resonance signals are quite different. Laser flash photolysis kinetic analysis of the reactions of the three forms of auracyanin with lumiflavin and flavin mononucleotide semiquinones indicates that the site of electron transfer is negatively charged and has an accessibility similar to that found in other blue copper proteins. Copper analysis indicates that all three proteins contain 1 mol of copper per mol of protein. All three auracyanins exhibit a midpoint redox potential of +240 mV. Light-induced absorbance changes and electron spin resonance signals suggest that auracyanin A may play a role in photosynthetic electron transfer. Kinetic data indicate that all three proteins can donate electrons to cytochrome c-554, the electron donor to the photosynthetic reaction center.

NASA Discipline Exobiology↗
DOE OSTI2020

Photoenzymatic Hydrogenation of Heteroaromatic Olefins Using ‘Ene’‐Reductases with Photoredox Catalysts

Abstract Flavin‐dependent ‘ene’‐reductases (EREDs) are highly selective catalysts for the asymmetric reduction of activated alkenes. This function is, however, limited to enones, enoates, and nitroalkenes using the native hydride transfer mechanism. Here we demonstrate that EREDs can reduce vinyl pyridines when irradiated with visible light in the presence of a photoredox catalyst. Experimental evidence suggests the reaction proceeds via a radical mechanism where the vinyl pyridine is reduced to the corresponding neutral benzylic radical in solution. DFT calculations reveal this radical to be “dynamically stable”, suggesting it is sufficiently long‐lived to diffuse into the enzyme active site for stereoselective hydrogen atom transfer. This reduction mechanism is distinct from the native one, highlighting the opportunity to expand the synthetic capabilities of existing enzyme platforms by exploiting new mechanistic models.

Nakano, Yuji↗
DOE OSTI2021

Engineering a Non‐Natural Photoenzyme for Improved Photon Efficiency**

Abstract Photoenzymes are biological catalysts that use light to convert starting materials into products. These catalysts require photon absorption for each turnover, making quantum efficiency an important optimization parameter. Flavin‐dependent “ene”‐reductases (EREDs) display latent photoenzymatic activity for synthetically valuable hydroalkylations; however, protein engineering has not been used to optimize this non‐natural function. We describe a protein engineering platform for the high throughput optimization of photoenzymes. A single round of engineering results in improved catalytic function toward the synthesis of γ, δ, ϵ‐lactams, and acyclic amides. Mechanistic studies show that key mutations can alter the enzyme's excited state dynamics, enhance its photon efficiency, and ultimately increase catalyst performance. Transient absorption spectroscopy reveals that engineered variants display dramatically decreased radical lifetimes, indicating an evolution toward a concerted mechanism.

Nicholls, Bryce T.↗
DOE OSTI2023

Ene Reductase Enabled Intramolecular β–C–H Functionalization of Substituted Cyclohexanones for Efficient Synthesis of Bridged Bicyclic Nitrogen Scaffolds

Herein we report that ene reductases (EREDs) can facilitate an unprecedented intramolecular β-C–H functionalization reaction for the synthesis of bridged bicyclic nitrogen heterocycles containing the 6-azabicyclo[3.2.1]octane scaffold. To streamline the synthesis of these privileged motifs, we developed a gram-scale one-pot chemoenzymatic cascade by combining iridium photocatalysis with EREDs, using readily available N-phenylglycines and cyclohexenones that can be obtained from biomass. Further derivatization using enzymatic or chemical methods can convert 6-azabicyclo[3.2.1]octan-3-one into 6-azabicyclo[3.2.1]octan-3α-ols, which can be potentially utilized for the synthesis of azaprophen and its analogues for drug discovery. Furthermore, mechanistic studies revealed the reaction requires oxygen, presumably to produce oxidized flavin, which can selectively dehydrogenate the 3-substituted cyclohexanone derivatives to form the α,β-unsaturated ketone, which subsequently undergoes spontaneous intramolecular aza-Michael addition under basic conditions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Ene Reductase Enabled Intramolecular β–C–H Functionalization of Substituted Cyclohexanones for Efficient Synthesis of Bridged Bicyclic Nitrogen Scaffolds

Herein we report that ene reductases (EREDs) can facilitate an unprecedented intramolecular β-C–H functionalization reaction for the synthesis of bridged bicyclic nitrogen heterocycles containing the 6-azabicyclo[3.2.1]octane scaffold. To streamline the synthesis of these privileged motifs, we developed a gram-scale one-pot chemoenzymatic cascade by combining iridium photocatalysis with EREDs, using readily available N-phenylglycines and cyclohexenones that can be obtained from biomass. Further derivatization using enzymatic or chemical methods can convert 6-azabicyclo[3.2.1]octan-3-one into 6-azabicyclo[3.2.1]octan-3α-ols, which can be potentially utilized for the synthesis of azaprophen and its analogues for drug discovery. Here, mechanistic studies revealed the reaction requires oxygen, presumably to produce oxidized flavin, which can selectively dehydrogenate the 3-substituted cyclohexanone derivatives to form the α,β-unsaturated ketone, which subsequently undergoes spontaneous intramolecular aza-Michael addition under basic conditions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Oleaginous Yeast Biology Elucidated With Comparative Transcriptomics

ABSTRACT Extremophilic yeasts have favorable metabolic and tolerance traits for biomanufacturing‐ like lipid biosynthesis, flavinogenesis, and halotolerance – yet the connection between these favorable phenotypes and strain genotype is not well understood. To this end, this study compares the phenotypes and gene expression patterns of biotechnologically relevant yeasts Yarrowia lipolytica , Debaryomyces hansenii , and Debaryomyces subglobosus grown under nitrogen starvation, iron starvation, and salt stress. To analyze the large data set across species and conditions, two approaches were used: a “network‐first” approach where a generalized metabolic network serves as a scaffold for mapping genes and a “cluster‐first” approach where unsupervised machine learning co‐expression analysis clusters genes. Both approaches provide insight into strain behavior. The network‐first approach corroborates that Yarrowia upregulates lipid biosynthesis during nitrogen starvation and provides new evidence that riboflavin overproduction in Debaryomyces yeasts is overflow metabolism that is routed to flavin cofactor production under salt stress. The cluster‐first approach does not rely on annotation; therefore, the coexpression analysis can identify known and novel genes involved in stress responses, mainly transcription factors and transporters. Therefore, this work links the genotype to the phenotype of biotechnologically relevant yeasts and demonstrates the utility of complementary computational approaches to gain insight from transcriptomics data across species and conditions.

Weintraub, Sarah J. [Department of Bioinformatics ↗
DOE OSTI2011

Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis

The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show that DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in L-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell. - Highlights: • The effect of interaction of nNOS with DLC1 has been debatable with contradicting reports in literature. • Purified DLC1 has no effect on electron transport between reductase and oxygenase domain of purified nNOS-CaM. • The NO release activity of nNOS was not altered by DLC1, supporting that DLC1 does not inhibit the enzyme. • These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Crystal structure of oleate hydratase from Stenotrophomonas sp. KCTC 12332 reveals conformational plasticity surrounding the FAD binding site

Highlights: • We determined oleate hydratase from Stenotrophomonas sp. KCTC 12332 in the apo state. • This shows a remarkable conformational change in the loop surrounding the bound FAD. • This change forces one of the important catalytic residues into position for catalysis. Unsaturated fatty acids are toxic to various bacteria, causing their death or growth inhibition. To prevent this toxicity, unsaturated fatty acids should be converted into saturated fatty acids via hydrogenation reaction, which is the complete reduction of double bonds on the carbon chain. In a recent report, we observed that Stenotrophomonas sp. KCTC 12332 exhibited a high biotransformation activity of oleic acid (OA) in 10-hydroxystearic acid and identified the gene encoding oleate hydratase (OhySt) by complete genomic analysis. In the present study, to further investigate the structural features of OhySt, the recombinant protein was expressed in Escherichia coli, and then purified and crystallized. Biochemical assay showed that OhySt produces 10-hydroxystearic acid in a flavin adenosine dinucleotide (FAD)-dependent manner, indicating that it requires FAD as a cofactor. The OhySt structure, which is determined in its apo state, allows for a structural comparison with the previously reported FAD bound structure of oleate hydratase. The comparison of structures indicates remarkable conformational change of the loop region surrounding the FAD molecule upon binding of FAD. This change forces one of the important catalytic residues into position for catalysis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Diphenyleneiodonium enhances oxidative stress and inhibits Japanese encephalitis virus induced autophagy and ER stress pathways

Highlights: 1.Diphenyleneiodonium (DPI) enhances ROS levels in Japanese encephalitis virus (JEV) infection. 2.DPI blocks autophagy and activation of ER stress pathways in JEV infected neuronal cells. 3.DPI inhibits JEV particle release. 4.During JEV infection autophagy and ER stress are likely to be modulated by DPI sensitive enzymes. Diphenyleneiodonium (DPI) and N-acetyl-l-cysteine (NAC), two widely used anti-oxidants, were employed to evaluate the role of oxidative stress in Japanese encephalitis virus (JEV) induced autophagy, stress responses and replication. DPI and NAC exerted opposite effects on ROS levels in JEV infected mouse neuronal cells (Neuro2a), mouse embryonic fibroblasts (MEFs) and human epithelial cells (HeLa). While NAC effectively quenched ROS, DPI enhanced ROS levels, suggesting that DPI induces oxidative stress in JEV infected cells. DPI treatment of JEV infected Neuro2a cells further blocked autophagy induction and activation of all three arms of the ER stress pathway, and, inhibited virus particle release. Autophagy induction in JEV infection has been previously shown to be linked to the activation of XBP1 and ATF6 ER stress sensors. Our data suggests that DPI mediated block of autophagy is a result of inhibition of ER stress responses and is not associated with an anti-oxidative effect. Since DPI has a wide inhibitory potential for all Flavin dependent enzymes, it is likely that the signalling pathways for ER stress and autophagy during JEV infection are modulated by DPI sensitive enzymes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Crystal structure of hypothetical protein PA4202 from Pseudomonas aeruginosa PAO1 in complex with nitroethane as a nitroalkane substrate

Highlights: • Crystal structure of PA4202 protein in complex with FMN and NE was determined. • A kinetic study reveals that the PA4202 enzyme may catalyze a neutral nitroalkane. • Our results provide the first structure-based substrate binding property of PA4202. Nitroalkane oxidase (NAO) and nitronate monooxygenase (NMO) are two different types of nitroalkane oxidizing flavoenzymes identified in nature. A previous study suggested that the hypothetical protein PA4202 from Pseudomonas aeruginosa PAO1 is NMO and utilizes only anionic nitronates. However, the structural similarity between the PA4202 protein and Streptomyces ansochromogenes NAO has motivated investigation for what features of the two enzymes differentiate between the NAO and NMO activities. Herein, we report the crystal structure of PA4202 in a ternary complex with a neutral nitroethane (NE) and flavin mononucleotide (FMN) cofactor to elucidate the substrate recognition mechanism using a site-directed mutagenesis. The ternary complex structure indicates that the NE is bound with an orientation, which is poised for the proton transfer to H183 (which is the essential first catalytic step with nitroalkanes), and subsequent reactions with FMN. Moreover, a kinetic study reveals that the catalytic reactions of the wild type and H183 mutants PA4202s with nitroalkane substrates may yield the products of hydrogen peroxide and nitrite that are specified to NAO, although they show a low catalytic efficiency. Our results provide the first structure-based molecular insight into the substrate binding property of the hypothetical protein PA4202, including the interactions with neutral nitroalkanes as the substrate.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Structures of glycolate oxidase from Nicotiana benthamiana reveal a conserved pH sensor affecting the binding of FMN

Highlights: • We solved the structures of apo- and FMN-bound GOX from Nicotiana benthamiana. • We reported the first crystal structure of GOX without FMN among all species. • We found that the binding of FMN induces a pronounced conformational change of GOX. • We found a conserved pH sensor that might directly regulate the binding of FMN. • We provided a feasible method to regulate the activity of GOX in patients and crops. Glycolate oxidase (GOX), a flavin mononucleotide (FMN)-dependent enzyme, modulates reactive oxygen species-mediated signal transduction in green plants. It has been a target protein for crop improvement because of performing a key step in photorespiration that causes the energy losses. In human, GOX is involved in the production of oxalate, which is a key metabolite in the formation of kidney stone. Here, we report the first apo-GOX structure and its complex structure with cofactor FMN from Nicotiana benthamiana by X-ray crystallography. The binding of FMN induces a pronounced conformational change of GOX tetramer. Interestingly, a conserved pH sensor found among different species might directly regulate the binding of FMN and the enzyme activity. Combined with enzymatic experiments and biophysical analyses, we provide new insights in the molecular mechanism of regulating GOX biological activity reversibly and new methods of agricultural production and clinical application.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

A hybrid cyt c maturation system enhances the bioelectrical performance of engineered Escherichia coli by improving the rate-limiting step

Bioelectronic devices can use electron flux to enable communication between biotic components and abiotic electrodes. We have modified Escherichia coli to electrically interact with electrodes by expressing the cytochrome c from Shewanella oneidensis MR-1. However, we observe inefficient electrical performance, which we hypothesize is due to the limited compatibility of the E. coli cytochrome c maturation (Ccm) systems with MR-1 cytochrome c. Here we test whether the bioelectronic performance of E. coli can be improved by constructing hybrid Ccm systems containing protein domains from both E. coli and S. oneidensis MR-1. The hybrid CcmH increased cytochrome c expression by increasing the abundance of CymA 60%, while only slightly changing the abundance of the other cytochromes c. Electrochemical measurements showed that the overall current from the hybrid ccm strain increased 121% relative to the wildtype ccm strain, with an electron flux per cell of 12.3 ± 0.3 fA·cell -1 . Additionally, the hybrid ccm strain doubled its electrical response with the addition of exogenous flavin, and quantitative analysis of this demonstrates CymA is the rate-limiting step in this electron conduit. These results demonstrate that this hybrid Ccm system can enhance the bioelectrical performance of the cyt c expressing E. coli, allowing the construction of more efficient bioelectronic devices.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase

Precursor molecules for biomass incorporation must be imported into cells and made available to the molecular machines that build the cell. Sulfur-containing macromolecules require that sulfur be in its S2- oxidation state before assimilation into amino acids, cofactors, and vitamins that are essential to organisms throughout the biosphere. In α-proteobacteria, NADPH-dependent assimilatory sulfite reductase (SiR) performs the final six-electron reduction of sulfur. SiR is a dodecameric oxidoreductase composed of an octameric flavoprotein reductase (SiRFP) and four hemoprotein metalloenzyme oxidases (SiRHPs). SiR performs the electron transfer reduction reaction to produce sulfide from sulfite through coordinated domain movements and subunit interactions without release of partially reduced intermediates. Efforts to understand the electron transfer mechanism responsible for SiR’s efficiency are confounded by structural heterogeneity arising from intrinsically disordered regions throughout its complex, including the flexible linker joining SiRFP’s flavin-binding domains. As a result, high-resolution structures of SiR dodecamer and its subcomplexes are unknown, leaving a gap in the fundamental understanding of how SiR performs this uniquely large-volume electron transfer reaction. In this work, we use deuterium labeling, in vitro reconstitution, analytical ultracentrifugation (AUC), small-angle neutron scattering (SANS), and neutron contrast variation (NCV) to observe the relative subunit positions within SiR’s higher-order assembly. AUC and SANS reveal SiR to be a flexible dodecamer and confirm the mismatched SiRFP and SiRHP subunit stoichiometry. NCV shows that the complex is asymmetric, with SiRHP on the periphery of the complex and the centers of mass between SiRFP and SiRHP components over 100 Å apart. SiRFP undergoes compaction upon assembly into SiR’s dodecamer and SiRHP adopts multiple positions in the complex. The resulting map of SiR’s higher-order structure supports a cis/trans mechanism for electron transfer between domains of reductase subunits as well as between tightly bound or transiently interacting reductase and oxidase subunits.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Structural studies of Neurospora crassa LPMO9D and redox partner CDHIIA using neutron crystallography and small-angle scattering

Sensitivity to hydrogen/deuterium and lack of observable radiation damage makes cold neutrons an ideal probe the structural studies of proteins with highly photosensitive groups such as the copper center of lytic polysaccharide monooxygenases (LPMOs) and flavin adenine dinucleotide (FAD) and heme redox cofactors of cellobiose dehydrogenases (CDHs). In this paper, neutron crystallography and small-angle neutron scattering are used to investigate Neurospora crassa LPMO9D (NcLPMO9D) and CDHIIA (NcCDHIIA), respectively. The presence of LPMO greatly enhances the efficiency of commercial glycoside hydrolase cocktails in the depolymerization of cellulose. LPMOs can receive electrons from CDHs to activate molecular dioxygen for the oxidation of cellulose resulting in chain cleavage and disruption of local crystallinity. Using neutron protein crystallography, the hydrogen/deuterium atoms of NcLPMO9D could be located throughout the structure. At the copper active site, the protonation states of the side chains of His1, His84, His157 and Tyr168, and the orientation of water molecules could be determined. Small-angle neutron scattering measurements provided low resolution models of NcCDHIIA with both the dehydrogenase and cytochrome domains in oxidized states that exhibited elongated conformations. Finally, this work demonstrates the suitability of neutron diffraction and scattering for characterizing enzymes critical to oxidative cellulose deconstruction.

59 BASIC BIOLOGICAL SCIENCES↗