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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 613 records · Page 34
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Evolutionary flexibility and rigidity in the bacterial methylerythritol phosphate (MEP) pathway

Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron–sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron–sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Photoenzymatic Enantioselective Intermolecular Radical Hydroalkylation: Experimental Crystal Structure Determination

Enzymes are increasingly explored for use in asymmetric synthesis, but their applications are generally limited by the reactions available to naturally occurring enzymes. Recently, interest in photocatalysis has spurred the discovery of novel reactivity from known enzymes. However, so far photoinduced enzymatic catalysis has not been used for the cross-coupling of two molecules. For example, the intermolecular coupling of alkenes with α-halo carbonyl compounds through a visible-light-induced radical hydroalkylation, which could provide access to important γ-chiral carbonyl compounds, has not yet been achieved by enzymes. The major challenges are the inherent poor photoreactivity of enzymes and the difficulty in achieving stereochemical control of the remote prochiral radical intermediate. Here we report a visible-light-induced intermolecular radical hydroalkylation of terminal alkenes that does not occur naturally, catalysed by an ‘ene’ reductase using readily available α-halo carbonyl compounds as reactants. This method provides an efficient approach to the synthesis of various carbonyl compounds bearing a γ-stereocentre with excellent yields and enantioselectivities (up to 99 percent yield with 99 percent enantiomeric excess), which otherwise are difficult to access using chemocatalysis. Mechanistic studies suggest that the formation of the complex of the substrates (α-halo carbonyl compounds) and the ‘ene’ reductase triggers the enantioselective photoinduced radical reaction. Our work further expands the reactivity repertoire of biocatalytic, synthetically useful asymmetric transformations by the merger of photocatalysis and enzyme catalysis.

Huang, Xiaoqiang↗
DOE OSTI2021

Plants with enhanced yield and methods of construction

Transgenic plants having enhanced yield and having enhanced seed yield are disclosed. The transgenic plants are transformed with a transgenic polynucleotide encoding one or more metabolic enzymes. The metabolic enzymes can be from any biological source. The transgenic polynucleotide(s) comprises a nucleic acid sequences encoding the metabolic enzymes under the control of functional plant promoters, the one or more metabolic enzymes are targeted to the plastids by the addition of plastid targeting signals. Optionally the functional plant promoters are seed specific promoters and the metabolic enzymes are targeted to the plastids by the addition of plastid targeting peptide heterologous to the metabolic enzymes. Methods of making the transgenic plants and transgenic polynucleotides are disclosed. The magnitude of the increases in seed yield achieved with these transgenic plants are simply unprecedented.

Malik, Meghna↗
DOE OSTI2024

Plants with enhanced yield and methods of construction

Transgenic plants having enhanced yield and having enhanced seed yield are disclosed. The transgenic plants are transformed with a transgenic polynucleotide encoding one or more metabolic enzymes. The metabolic enzymes can be from any biological source. The transgenic polynucleotide(s) comprises a nucleic acid sequences encoding the metabolic enzymes under the control of functional plant promoters, the one or more metabolic enzymes are targeted to the plastids by the addition of plastid targeting signals. Optionally the functional plant promoters are seed specific promoters and the metabolic enzymes are targeted to the plastids by the addition of plastid targeting peptide heterologous to the metabolic enzymes. Methods of making the transgenic plants and transgenic polynucleotides are disclosed. The magnitude of the increases in seed yield achieved with these transgenic plants are unprecedented.

Malik, Meghna↗
DOE OSTI2026

Engineering and Application of a Thermostable MHETase for PET Depolymerization

Enzymatic hydrolysis of poly(ethylene terephthalate) (PET) releases mono(2-hydroxyethyl) terephthalate (MHET) as a major product, the accumulation of which can prolong reactor residence times and complicate downstream monomer separations. The use of a MHETase enzyme can enable MHET hydrolysis to the monomers, terephthalic acid and ethylene glycol, but industrial PETases typically operate at thermophilic temperatures and the well-known MHETase from Ideonella sakaiensis is a mesophilic enzyme, thus warranting the development of thermophilic MHETases. Here, we characterize thermostable MHET-active enzymes from a natural diversity screen by applying a hidden Markov model based on the previously reported, archaeal ferulic acid esterase, PET46. We identified enzymes with higher thermostability than PET46 and quantified their MHETase activity in reactions at 70 °C. The crystal structure of MHT077, the homologue with the highest MHETase activity and an apparent melting temperature (T m,app ) of 94.6 °C, informed site saturation mutagenesis in the active site and lid-domain interface. MHT077 exhibited a ∼100-fold slower unfolding rate at 65 °C than PET46, indicating substantially greater kinetic stability. In parallel, we applied evolution-informed design, a probabilistic model that leverages coevolutionary patterns in large multiple sequence alignments, to improve the activity and thermostability of five ferulic acid esterases. One design, EV-MHT043–5 was identified with a comparable thermostability (T m,app = 96.1 °C) and a 3-fold improvement in its MHETase activity relative to the wildtype enzyme, MHT043. Combination variants of beneficial mutations were screened and afforded a variant, MHT077 LFK , which reduced MHET accumulation in bioreactor experiments with postconsumer PET waste. Overall, this study expands the known MHET-hydrolyzing protein scaffolds available for enzymatic PET recycling.

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

Differential Organic Carbon Mineralization Responses to Soil Moisture in Three Different Soil Orders Under Mixed Forested System: Supporting Data

This data contains data from 90-day long incubation study which aimed to look at the soil moisture-texture relationship on soil organic carbon (SOC) cycling. Soils were collected from three distinct soil textures from mixed forests in 2017: sandy (Georgia, 2017-05-01), loamy (Missouri, 2017-06-14) and clayey (Texas, December 2017) were incubated at different soil moisture levels (air-dried, 25% water holding capacity (WHC), 50% WHC, 100% WHC and 175% WHC) at room temperature for a period of 90 days. Files contain microbial respiration, active and slow SOC pools, and their respective mineralization rates, extractable organic carbon (C), and C-acquiring extracellular enzymes. Findings from these data were used in Singh et al. (2021). This study aimed to examine the interactive effect of soil moisture and texture on SOC mineralization. Soil samples of three distinct textures (sandy, loamy, and clayey) were collected from mixed forests of Georgia, Missouri, and Texas, respectively. Soil cores of 5 cm diameter were collected from numerous random locations at each site from 0-15 cm depth after scraping the litter layer and mixed thoroughly to obtain a composite sample per site. Three additional soil cores were collected to determine the WHC using pressure plate extractors. Soil samples were composited, and triplicate soil samples were incubated in mason jars for a period of 90 days at room temperature under different moisture regimes: air dried, 25% WHC, 50% WHC, at WHC and 100% saturation. Soil respiration was measured weekly, and destructive sampling was conducted at 1, 15, 60, and 90 days to determine extractable organic C, C acquiring enzyme activity, and active and slow SOC pools with their respective mineralization rates. The C acquiring enzyme activity was the total activity of α-glucosidase, β-glucosidase, cellobiohydrolase, and β-xylosidase enzymes. Gas samples for microbial respiration measurements were collected from headspace of incubation jars through the sampling ports on the lids and then analyzed using a Shimadzu Gas Chromatograph (GC-2014). Prior to sampling, the vials were evacuated. Blank correction was also done by collecting gas samples from empty incubation jars. Double pool exponential decay model was used in SigmaPlot to determine the active and slow SOC pools and their mineralization rates (Farrar et al., 2012; Jagadamma et al., 2014). The C-acquiring extracellular enzymes were measured using the microplate method by German et al., (2011). Microbial community structure was determined using the phospholipid fatty acid (PLFA) and neutral lipid fatty acid (NLFA) analyses (Buyer and Sasser, 2012). This dataset has seven data files provided in comma-separate (*.csv) format. Additional metadata are provided: seven data dictionaries and a file-level metadata file in comma separate (*.csv) format and a user guide in PDF (*.pdf) format.

Carbon acquiring enzyme activity↗
DOE OSTI2021

A Multiomic Approach to Understand How Pleurotus eryngii Transforms Non-Woody Lignocellulosic Material

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Protein–Protein Complex Stability Controls Substrate Scope in a β‐Ketoacyl‐ACP Reductase Specific for Medium Chains

Assembly-line enzymes carry out multistep synthesis of important metabolites by using acyl carrier proteins (ACPs) to shuttle intermediates along defined sequences of active sites. Despite longstanding interest in reprogramming these systems for metabolic engineering and biosynthetic chemistry, the mechanisms underlying their reaction order remain poorly understood and difficult to control. In this work, we describe a β-ketoacyl-ACP reductase from Pseudomonas putida (PpFabG4) with an unusual selectivity for medium chains and use it to explore the molecular basis of substrate specificity in enzymes that pull intermediates from fatty acid synthesis, a common route to specialized products. X-ray crystallography shows no obvious barriers to short-chain binding. Molecular simulations and supporting mutational analyses indicate that substrate preference arises instead from a weak enzyme–ACP interaction that is stabilized by medium acyl chains but not by short chains. Indeed, mutations that strengthen this interaction for PpFabG4 or weaken it for EcFabG, an Escherichia coli β-ketoacyl-ACP reductase with a broad substrate specificity, can enhance or reduce activity on short-chain substrates by over 100-fold. Our findings show how the stability of enzyme-ACP interactions can control substrate scope in promiscuous enzymes and guide the exchange of intermediates between (and within) assembly-line systems.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Continuous Directed Evolution of a Short-Lived Plant Histidinol Dehydrogenase

Enzyme protein turnover accounts for about half the maintenance energy budget in plants. Slowing turnover i.e., extending the effective working life (Catalytic Cycles till Replacement, CCR) of short-lived enzymes is thus a rational strategy to conserve energy and carbon and raise crop productivity. Arabidopsis histidinol dehydrogenase (HDH) is a short-lived enzyme that can sustain lifeshortening damage from its aminoaldehyde reaction intermediate. We used the yeast OrthoRep continuous directed evolution system in a his4 Δ strain to raise cumulative HDH function and, by proxy, lifespan as functional enzymes, by selecting for growth rate while tapering histidinol concentration and escalating that of the inhibitor histamine. Improved HDH variants carried diverse nonsynonymous mutations and ranged 20-fold in level. Improved HDH performance was associated with higher HDH abundance in some cases and with greater catalytic efficiency or histamine resistance in others. These findings indicate that OrthoRep-based directed evolution can extend enzyme working life in vivo in addition to, as expected, altering kinetic properties.

directed evolution↗
DOE OSTI2023

Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography

Pyridoxal 5’-phosphate (PLP)-dependent enzymes utilize a vitamin B 6 -derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of small-molecule antimicrobials and anticancer therapeutics, while others have been used to synthesize pharmaceutical building blocks. Understanding PLP-dependent catalysis and the reaction specificity is crucial to advance structure-assisted drug design and enzyme engineering. Here we report the direct determination of the protonation states in the active site of Thermus thermophilus SHMT (TthSHMT) in the internal aldimine state using room-temperature joint X-ray/neutron crystallography. Conserved active site architecture of the model enzyme TthSHMT and of human mitochondrial SHMT (hSHMT2) were compared by obtaining a room-temperature X-ray structure of hSHMT2, suggesting identical protonation states in the human enzyme. The amino acid substrate serine pathway through the TthSHMT active site cavity was tracked, revealing the peripheral and cationic binding sites that correspond to the pre-Michaelis and pseudo-Michaelis complexes, respectively. At the peripheral binding site, the substrate is bound in the zwitterionic form. By analyzing the observed protonation states, Glu53, but not His residues, is proposed as the general base catalyst, orchestrating the retro-aldol transformation of L-serine into glycine.

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

Bacterial microcompartments as a next-generation metabolic engineering tool: utilizing nature's solution for confining challenging catabolic pathways

Advancements in synthetic biology have facilitated the incorporation of heterologous metabolic pathways into various bacterial chassis, leading to the synthesis of targeted bioproducts. However, total output from heterologous production pathways can suffer from low flux, enzyme promiscuity, formation of toxic intermediates, or intermediate loss to competing reactions, which ultimately hinder their full potential. The self-assembling, easy-to-modify, protein-based bacterial microcompartments (BMCs) offer a sophisticated way to overcome these obstacles by acting as an autonomous catalytic module decoupled from the cell's regulatory and metabolic networks. More than a decade of fundamental research on various types of BMCs, particularly structural studies of shells and their self-assembly, the recruitment of enzymes to BMC shell scaffolds, and the involvement of ancillary proteins such as transporters, regulators, and activating enzymes in the integration of BMCs into the cell's metabolism, has significantly moved the field forward. These advances have enabled bioengineers to design synthetic multi-enzyme BMCs to promote ethanol or hydrogen production, increase cellular polyphosphate levels, and convert glycerol to propanediol or formate to pyruvate. These pioneering efforts demonstrate the enormous potential of synthetic BMCs to encapsulate non-native multi-enzyme biochemical pathways for the synthesis of high-value products.

59 BASIC BIOLOGICAL 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 OSTI2025

Unravelling the Glycan Code: Molecular Dynamics and Quantum Chemistry Reveal How O-Glycan Functional Groups Govern OgpA Selectivity in Mucin Degradation by Akkermansia muciniphila

Mucins, heavily O-glycosylated glycoproteins, are a key component of mucus, and certain gut microbiota, including Akkermansia muciniphila , can utilise mucin glycans as a carbon source. Akkermansia muciniphila produces the O-glycopeptidase enzyme OgpA, which cleaves peptide bonds at the N-terminus of serine (Ser) or threonine (Thr) residues carrying O-glycan substitutions, with selectivity influenced by the O-glycan functional groups. Using molecular dynamics (MD) simulations and quantum chemistry calculations, we explored how different O-glycan groups affect OgpA's selectivity. Our results show that peptides bind to the enzyme via hydrogen bonds, π–π interactions, van der Waals forces and electrostatic interactions, with key residues, including Tyr90, Val138, Gly176, Tyr210 and Glu91, playing important roles. The primary determinant of selectivity is the interaction between the peptide's functional group and the enzyme's binding cavity, while peptide–enzyme interface interactions are secondary. Quantum chemistry calculations reveal that OgpA prefers peptides with a lower electrophilic character. This study provides new insights into mucin degradation by gut microbiota enzymes, advancing our understanding of this critical biological process.

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

MOFLUX Intensified Soil Moisture Extremes Decrease Soil Organic Carbon Decomposition: Modeling Archive

This Modeling Archive is in support the publication “Intensified Soil Moisture Extremes Decrease Soil Organic Carbon Decomposition: A Mechanistic Modeling Analysis” (Liang et al., 2021). Here we provide model code, inputs, outputs and evaluation datasets for the Microbial ENzyme Decomposition (MEND) model for the Missouri Ozarks AmeriFlux eddy covariance measurement site (MOFLUX) near Ashland, Missouri USA. The MEND model was developed with explicit representation of microbial and enzyme pools to mechanistically simulate the role of microbial organisms and extracellular enzymes in soil organic carbon (SOC) decomposition. Long-term SOC dynamics under intensified moisture extremes are studied using the MEND model that is parameterized with 11 years of measurements from the MOFLUX forest. The model explicitly represents microbial dormancy and resuscitation, different types of SOC-degrading enzymes, and how they vary with changes in soil moisture (Wang et al. 2015, 2019). A combination of two levels of frequency and severity of soil moisture, as well as a control with normal interannual variability, are used to simulate a range of moisture scenarios over 100 years. The code of Microbial-ENzyme Decomposition (MEND) as well as the input and output data are included in the archive. A user’s manual (MEND_Readme.pdf) is included with instructions for compiling and running the model to simulate soil organic carbon decomposition under various moisture scenarios. This dataset contains the modelling archive contained within a compressed (*.zip) file, a file-level metadata file in comma separate (*.csv) format, and two instructional files in PDF (*.pdf) format.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Biocatalytic Membranes for Carbon Capture and Utilization

Innovative carbon capture technologies that capture CO 2 from large point sources and directly from air are urgently needed to combat the climate crisis. Likewise, corresponding technologies are needed to convert this captured CO 2 into valuable chemical feedstocks and products that replace current fossil-based materials to close the loop in creating viable pathways for a renewable economy. Biocatalytic membranes that combine high reaction rates and enzyme selectivity with modularity, scalability, and membrane compactness show promise for both CO 2 capture and utilization. This review presents a systematic examination of technologies under development for CO 2 capture and utilization that employ both enzymes and membranes. CO 2 capture membranes are categorized by their mode of action as CO 2 separation membranes, including mixed matrix membranes (MMM) and liquid membranes (LM), or as CO 2 gas–liquid membrane contactors (GLMC). Because they selectively catalyze molecular reactions involving CO 2 , the two main classes of enzymes used for enhancing membrane function are carbonic anhydrase (CA) and formate dehydrogenase (FDH). Small organic molecules designed to mimic CA enzyme active sites are also being developed. CO 2 conversion membranes are described according to membrane functionality, the location of enzymes relative to the membrane, which includes different immobilization strategies, and regeneration methods for cofactors. Parameters crucial for the performance of these hybrid systems are discussed with tabulated examples. Progress and challenges are discussed, and perspectives on future research directions are provided.

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

Utilization of protein film electrochemistry to characterize the mechanisms imparting aerotolerance and bidirectionality in soluble, multimeric [NiFe]-hydrogenases (Final Report for Grant No. DE-SC0008074)

The overall goal of this project was to better understand how structural features control functional aspects of enzymes involved in catalysis in which a substrate is oxidized or reduced. Two types of model enzymes were explored: the soluble [NiFe]-hydrogenase from Pyrococcus furiousus and the thiosulfate reductase from Pyrobaculum aerophilum. Both are models for complex, multielectron transformations such as nitrogen and carbon dioxide reduction, and parts of important biogeochemical cycles. In this project, electrochemical methods were used to define the activities of these enzymes and targeted variants designed to modulate activity. We showed that the catalytic activity of the soluble hydrogenase from Pyrococcus furiosus can be modulated by removing large parts of the enzyme far from the active site. This surprising result suggests that interactions between functional centers far apart within the enzyme may be essential for controlling and tuning activity. We also showed that the catalytic activity of thiosulfate reductase from Pyrobaculum aerophilum can be modulated by changing the metal at the active site to be either molybdenum or tungsten. These results contribute to our understanding of how the protein environment can tune the reactivity of an embedded metal, catalytic center. The results were and are being published in peer-reviewed, scientific publications.

08 HYDROGEN↗
DOE OSTI2021

Characterization of protein–ligand binding interactions of enoyl-ACP reductase ( FabI ) by native MS reveals allosteric effects of coenzymes and the inhibitor triclosan

The enzyme enoyl-ACP reductase (also called FabI in bacteria) is an essential member of the fatty acid synthase II pathway in plants and bacteria. This enzyme is the target of the antibacterial drug triclosan and has been the subject of extensive studies for the past 20 years. Despite the large number of reports describing the biochemistry of this enzyme, there have been no studies that provided direct observation of the protein and its various ligands. In this paper we describe the use of native MS to characterize the protein–ligand interactions of FabI with its coenzymes NAD + and NADH and with the inhibitor triclosan. Measurements of the gas-phase affinities of the enzyme for these ligands yielded values that are in close agreement with solution-phase affinity measurements. Additionally, FabI is a homotetramer and we were able to measure the affinity of each subunit for each coenzyme, which revealed that both coenzymes exhibit a positive homotropic allosteric effect. An allosteric effect was also observed in association with the inhibitor triclosan. These observations provide new insights into this well-studied enzyme and suggest that there may still be gaps in the existing mechanistic models that explain FabI inhibition.

59 BASIC BIOLOGICAL SCIENCES↗