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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 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Development of energetic and enzymatic limitations on microbial carbon cycling in soils

Soil organic carbon (SOC) constitutes an important reservoir in the global carbon cycle that is vulnerable to transformation and loss from land use and climate change. Anoxic conditions protect SOC from microbial degradation through limiting the energetics of respiration and inhibiting extracellular oxidative enzymes. Given growing evidence of prevalent anaerobic microsites in upland soils, we designed an experiment testing the development of dissolved organic carbon (DOC) signatures of energetic and enzymatic limitations on microbial carbon utilization across simulated soil aggregates or peds. Reactors comprised a soil column “aggregate” underlying an advective “macropore” channel. Soils received downward diffusive inputs of aerated porewater media with added nitrate, sulfate, or no amendment—where native ferrihydrite served as dominant anaerobic terminal electron acceptor (TEA). After 40 days, added nitrate resulted in highest bulk respiration and DOC production while sulfate did not differ from the control. Nominal oxidation state of carbon (NOSC) was higher (more favorable) with added TEAs at soil surfaces and decreased with depth, while NOSC in the non-amended soil remained lower and constant with depth. DOC generally increased with depth, which along with decreasing NOSC values indicates joint electron-donor and acceptor control over respiration energetics. Of all organic compound classes, only the relative abundance of phenolics increased between 0 and 0.5 cm depth, which aligns with the oxic-anoxic transition and suggests oxidative enzyme inhibition. Furthermore, our results suggest that oxygen limitation within upland soil aggregates may preserve SOC via both energetic and enzymatic C protection mechanisms, which are vulnerable upon exposure to oxygen.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Quaternary Charge-Transfer Complex Enables Photoenzymatic Intermolecular Hydroalkylation of Olefins

Intermolecular C–C bond-forming reactions are underdeveloped transformations in the field of biocatalysis. Here we report a photoenzymatic intermolecular hydroalkylation of olefins catalyzed by flavin-dependent ‘ene’-reductases. Radical initiation occurs via photoexcitation of a rare high-order enzyme-templated charge-transfer complex that forms between an alkene, α-chloroamide, and flavin hydroquinone. This unique mechanism ensures that radical formation only occurs when both substrates are present within the protein active site. Here, this active site can control the radical terminating hydrogen atom transfer, enabling the synthesis of enantioenriched γ-stereogenic amides. This work highlights the potential for photoenzymatic catalysis to enable new biocatalytic transformations via previously unknown electron transfer mechanisms.

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

Ubiquitin-like conjugation by bacterial cGAS enhances anti-phage defence

Abstract cGAS is an evolutionarily conserved enzyme that has a pivotal role in immune defence against infection 1–3 . In vertebrate animals, cGAS is activated by DNA to produce cyclic GMP–AMP (cGAMP) 4,5 , which leads to the expression of antimicrobial genes 6,7 . In bacteria, cyclic dinucleotide (CDN)-based anti-phage signalling systems (CBASS) have been discovered 8–11 . These systems are composed of cGAS-like enzymes and various effector proteins that kill bacteria on phage infection, thereby stopping phage spread. Of the CBASS systems reported, approximately 39% contain Cap2 and Cap3 , which encode proteins with homology to ubiquitin conjugating (E1/E2) and deconjugating enzymes, respectively 8,12 . Although these proteins are required to prevent infection of some bacteriophages 8 , the mechanism by which the enzymatic activities exert an anti-phage effect is unknown. Here we show that Cap2 forms a thioester bond with the C-terminal glycine of cGAS and promotes conjugation of cGAS to target proteins in a process that resembles ubiquitin conjugation. The covalent conjugation of cGAS increases the production of cGAMP. Using a genetic screen, we found that the phage protein Vs.4 antagonized cGAS signalling by binding tightly to cGAMP (dissociation constant of approximately 30 nM) and sequestering it. A crystal structure of Vs.4 bound to cGAMP showed that Vs.4 formed a hexamer that was bound to three molecules of cGAMP. These results reveal a ubiquitin-like conjugation mechanism that regulates cGAS activity in bacteria and illustrates an arms race between bacteria and viruses through controlling CDN levels.

Science & Technology - Other Topics↗
DOE OSTI2024

Effects of SARS-CoV-2 Main Protease Mutations at Positions L50, E166, and L167 Rendering Resistance to Covalent and Noncovalent Inhibitors

SARS-CoV-2 propagation under nirmatrelvir and ensitrelvir pressure selects for main protease (MPro) drug-resistant mutations E166V (DRM2), L50F/E166V (DRM3), E166A/L167F (DRM4), and L50F/E166A/L167F (DRM5). DRM2-DRM5 undergoes N-terminal autoprocessing to produce mature MPro with dimer dissociation constants (K dimer ) 2–3 times larger than that of the wildtype. Co-selection of L50F restores catalytic activity of DRM2 and DRM4 from ~10 to 30%, relative to that of the wild-type enzyme, without altering K dimer . Binding affinities and thermodynamic profiles that parallel the drug selection pressure, exhibiting significant decreases in affinity through entropy/enthalpy compensation, were compared with GC373. Reorganization of the active sites due to mutations observed in the inhibitor-free DRM3 and DRM4 structures as compared to MPro WT may account for the reduced binding affinities, although DRM2 and DRM3 complexes with ensitrelvir are almost identical to MPro WT -ensitrelvir. In conclusion, chemical reactivity changes of the mutant active sites due to differences in electrostatic and protein dynamics effects likely contribute to losses in binding affinities.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Methods and compositions to increase the rate of ligation reactions catalyzed by a sortase

In various embodiments methods and compositions to increase the rate and/or activity of ligation reactions catalyzed by a sortase. In certain embodiments reagents are provided that comprises, inter alia, a polypeptide comprising an amino terminal polyglycine sequence comprising at least three contiguous Gly residues (e.g., a triglycine sequence, a tetraglycine sequence, a pentaglycine sequence, etc.) followed by a peptide that is to be ligated to a moiety followed by a sequence comprising the catalytic domain of a Sortase A enzyme.

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

Structural insights into strigolactone catabolism by carboxylesterases reveal a conserved conformational regulation

Phytohormone levels are regulated through specialized enzymes, participating not only in their biosynthesis but also in post-signaling processes for signal inactivation and cue depletion. Arabidopsis thaliana (At) carboxylesterase 15 (CXE15) and carboxylesterase 20 (CXE20) have been shown to deplete strigolactones (SLs) that coordinate various growth and developmental processes and function as signaling molecules in the rhizosphere. Here, we elucidate the X-ray crystal structures of AtCXE15 (both apo and SL intermediate bound) and AtCXE20, revealing insights into the mechanisms of SL binding and catabolism. The N-terminal regions of CXE15 and CXE20 exhibit distinct secondary structures, with CXE15 characterized by an alpha helix and CXE20 by an alpha/beta fold. These structural differences play pivotal roles in regulating variable SL hydrolysis rates. Our findings, both in vitro and in planta, indicate that a transition of the N-terminal helix domain of CXE15 between open and closed forms facilitates robust SL hydrolysis. The results not only illuminate the distinctive process of phytohormone breakdown but also uncover a molecular architecture and mode of plasticity within a specific class of carboxylesterases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Enzymatic carbon–fluorine bond cleavage by human gut microbes

Fluorinated compounds are used for agrochemical, pharmaceutical, and numerous industrial applications, resulting in global contamination. In many molecules, fluorine is incorporated to enhance the half-life and improve bioavailability. Fluorinated compounds enter the human body through food, water, and xenobiotics including pharmaceuticals, exposing gut microbes to these substances. The human gut microbiota is known for its xenobiotic biotransformation capabilities, but it was not previously known whether gut microbial enzymes could break carbon-fluorine bonds, potentially altering the toxicity of these compounds. Here, through the development of a rapid, miniaturized fluoride detection assay for whole-cell screening, we identified active gut microbial defluorinases. We biochemically characterized enzymes from diverse human gut microbial classes including Clostridia, Bacilli, and Coriobacteriia, with the capacity to hydrolyze (di)fluorinated organic acids and a fluorinated amino acid. Whole-protein alanine scanning, molecular dynamics simulations, and chimeric protein design enabled the identification of a disordered C-terminal protein segment involved in defluorination activity. Domain swapping exclusively of the C-terminus conferred defluorination activity to a nondefluorinating dehalogenase. To advance our understanding of the structural and sequence differences between defluorinating and nondefluorinating dehalogenases, we trained machine learning models which identified protein termini as important features. Models trained on 41-amino acid segments from protein C termini alone predicted defluorination activity with 83% accuracy (compared to 95% accuracy based on full-length protein features). This work is relevant for therapeutic interventions and environmental and human health by uncovering specificity-determining signatures of fluorine biochemistry from the gut microbiome.

Probst, Silke I↗
DOE OSTI2023

Facile and dynamic cleavage of every iron–sulfide bond in cuboidal iron–sulfur clusters

Nature employs weak-field metalloclusters to support a wide range of biological processes. The most ubiquitous metalloclusters are the cuboidal Fe–S clusters, which are comprised of Fe sites with locally high-spin electronic configurations. Such configurations enhance rates of ligand exchange and imbue the clusters with a degree of structural plasticity that is increasingly thought to be functionally relevant. Here, we examine this phenomenon using isotope tracing experiments. Specifically, we demonstrate that synthetic [Fe 4 S 4 ] and [MoFe 3 S 4 ] clusters exchange their Fe atoms with Fe 2+ ions dissolved in solution, a process that involves the reversible cleavage and reformation of every Fe–S bond in the cluster core. This exchange is facile—in most cases occurring at room temperature on the timescale of minutes—and documented over a range of cluster core oxidation states and terminal ligation patterns. In addition to suggesting a highly dynamic picture of cluster structure, these results provide a method for isotopically labeling pre-formed clusters with spin-active nuclei, such as 57 Fe. Such a protocol is demonstrated for the radical S -adenosyl- l -methionine enzyme, RlmN.

Science & Technology - Other Topics↗
DOE OSTI2021

Physiological, genomic, and sulfur isotopic characterization of methanol metabolism by Desulfovibrio carbinolicus

Methanol is often considered as a non-competitive substrate for methanogenic archaea, but an increasing number of sulfate-reducing microorganisms (SRMs) have been reported to be capable of respiring with methanol as an electron donor. A better understanding of the fate of methanol in natural or artificial anaerobic systems thus requires knowledge of the methanol dissimilation by SRMs. In this study, we describe the growth kinetics and sulfur isotope effects of Desulfovibrio carbinolicus, a methanol-oxidizing sulfate-reducing deltaproteobacterium, together with its genome sequence and annotation. D. carbinolicus can grow with a series of alcohols from methanol to butanol. Compared to longer-chain alcohols, however, specific growth and respiration rates decrease by several fold with methanol as an electron donor. Larger sulfur isotope fractionation accompanies slowed growth kinetics, indicating low chemical potential at terminal reductive steps of respiration. In a medium containing both ethanol and methanol, D. carbinolicus does not consume methanol even after the cessation of growth on ethanol. Among the two known methanol dissimilatory systems, the genome of D. carbinolicus contains the genes coding for alcohol dehydrogenase but lacks enzymes analogous to methanol methyltransferase. We analyzed the genomes of 52 additional species of sulfate-reducing bacteria that have been tested for methanol oxidation. There is no apparent relationship between phylogeny and methanol metabolizing capacity, but most gram-negative methanol oxidizers grow poorly, and none carry homologs for methyltransferase (mtaB). Although the amount of available data is limited, it is notable that more than half of the known gram-positive methanol oxidizers have both enzymatic systems, showing enhanced growth relative to the SRMs containing only alcohol dehydrogenase genes. Thus, physiological, genomic, and sulfur isotopic results suggest that D. carbinolicus and close relatives have the ability to metabolize methanol but likely play a limited role in methanol degradation in most natural environments.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Direct interaction of DNA repair protein tyrosyl DNA phosphodiesterase 1 and the DNA ligase III catalytic domain is regulated by phosphorylation of its flexible N-terminus

Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3'-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped by either DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA-binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII 170-755 , as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small-angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in nonmalignant and cancer cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Identification of a carbonic anhydrase–Rubisco complex within the alpha-carboxysome

Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO 2 fixation—Rubisco and carbonic anhydrase—and are the centerpiece of the bacterial CO 2 concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO 2 by carbonic anhydrase, producing a high CO 2 concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the α-carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how the carbonic anhydrase, CsoSCA, is incorporated into the α-carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into α-carboxysomes from Halothiobacillus neapolitanus . We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 Å single-particle cryoelectron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCA's binding site overlaps with that of CsoS2, but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of carboxysome biogenesis and highlight the importance of Rubisco, not only as an enzyme but also as a central hub for mediating assembly through protein interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme’s active site. Our findings provide new insights for the development of uracil scaffold-based drugs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The Bacteroidetes Aequorivita sp. and Kaistella jeonii Produce Promiscuous Esterases With PET-Hydrolyzing Activity

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil ® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the Is PETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as Is PETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Novel Microbial Based Enzymatic CO 2 Fixation Mechanisms

The broad, long term goal of the research is to provide insight into the mechanism of novel carboxylation reactions catalyzed by enzymes involved in bacterial alkene, ketone, and epoxide metabolism. Novel convergent pathways in the model microorganism Xanthobacter autotrophicus have been described in which epoxides produced by alkene oxidation and ketones produced by the oxidation of alcohols are further carboxylated and utilized in energy yielding pathways. Both alkene/epoxide and alcohol/ketone pathways involve distinct carboxylases with unique molecular properties and cofactor requirements that represent alternative mechanisms of CO 2 fixation. The main focus and aims of this project is to provide structural basis for the mechanistic understanding of the reactions catalyzed by 2-ketopropyl coenzyme M oxidoreductase / carboxylase and acetone carboxylase, the terminal steps in the convergent pathways of alkene/epoxide and alcohol ketone metabolism in X. autotrophicus strain Py2. Interestingly, coenzyme M (CoM), the simplest known organic coenzyme, has been shown to have a central role in the pathway of alkene/epoxide carboxylation. Prior to this discovery CoM was thought only to be involved in methanogenesis in archaea. In addition to the carboxylation mechanisms, we are examining the enzymology of the biosynthesis of CoM in bacteria and we have already determined that it is biochemically distinct from that of CoM synthesized by methanogens. The proposed studies will reveal new and unique insights into CO 2 fixation pathways and carboxylation chemistry. We are utilizing a multidisciplinary approach involving kinetic studies, site-specific amino acid substitution studies, and the determination of high-resolution structures of enzymes in the presence of substrate analogs and mechanism base inhibitors to ascertain information concerning the biochemical mechanisms of enzyme catalyzed reactions. Within the context of the mission of the Department of Energy and the core activities of the Energy Biosciences, the results obtained in the study will reveal new insights into these novel carboxylation reactions and will provide the basis for the comparison of the mechanism of these interesting enzymes to other well-characterized biological mechanisms for CO 2 fixation and carboxylation. Knowledge concerning the mechanism of these enzymes provides the basis for an increased fundamental understanding of carboxylation chemistry that could contribute to future mechanisms for CO 2 capture and fixation and, in turn, to mitigating the effects of increasing concentrations of CO 2 on global climate change.

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

Development of the high-productivity marine microalga, Picochlorum renovo , as a photosynthetic protein secretion platform

Microalgae are remarkable in their ability to convert CO 2 and sunlight into renewable biomass and bioproducts. In this work, we have established a novel photosynthetic protein production platform via computational prediction and genetic engineering using native microalgal protein secretory signal peptides to achieve functional secretion of the fluorescent protein mCherry. Importantly, this work was conducted in the recently characterized alga Picochlorum renovo, an alga of industrial interest due to its rapid growth rate, tolerance to both high temperature and salinity, and genetically tractable nuclear and chloroplast genomes. Genomic queries allowed the identification of native secretory signal peptides, which were N-terminally fused to mCherry allowing for secretion into the culture supernatant. Further characterization revealed no impact on fitness, a production rate of 0.19 mg/L/day, and titer of 0.37 mg/L of transgenic mCherry protein in culture supernatant. These findings lay the foundation for applied genetic engineering approaches that could enable CO 2 -sequestering, sustainable photoproduction of industrially relevant enzymes at low cost.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

A characterization of recombinant Arabidopsis FRIABLE1 (FRB1) reveals robust rhamnogalacturonan-I rhamnosyltransferase activity and critical catalytic residues

Plant cell walls are glycan-rich extracellular matrices that fundamentally impact essential cellular processes, such as growth, adhesion, and cell shape acquisition. Understanding plant cell wall glycans requires the identification and characterization of the biosynthetic enzymes that produce these polymers. Most successful in vitro protein expression studies of plant cell wall glycosyltransferases have relied on insect, fungal/yeast, or human cell expression systems, whereas prokaryotic expression systems have been generally unsuccessful. Here, we show that Arabidopsis FRIABLE1 (FRB1)/rhamnogalacturonan-I rhamnosyltransferase 8 (RRT8) can be produced in Escherichia coli RosettaGami2 cells as N-terminal maltose-binding protein fusion proteins containing C-terminal 6X-His-tags. We also report the catalytic constants of FRB1/RRT8 with apparent K M and K cat values of 226 μM and 33 min -1 for UDP-Rhamnose and 117 μM and 28.7 min -1 for rhamnogalacturonan-I (RG-I), respectively. We examine the catalytic activities of mutated FRB1/RRT8 proteins based on an AlphaFold 3-generated FRB1/RRT8 protein structural model with a virtually docked UDP-Rha donor. Enzymatic characterization of the mutated and wildtype FRB1/RRT8 protein confirmed that mutation of predicted catalytic site amino acid residues resulted in a 20-fold reduction in RRT activity. FRB1 also robustly polymerizes RG-I in combination with RG-I galacturonosyltransferase 1. These results show how a robust E. coli expression system combined with artificial intelligence tools can be used to increase understanding of plant cell wall glycosyltransferase structure and function.

glycosyltransferase↗
DOE OSTI2022

Domain architecture and catalysis of the Staphylococcus aureus fatty acid kinase

Fatty acid kinase (Fak) is a two-component enzyme that generates acyl-phosphate for phospholipid synthesis. Fak consists of a kinase domain protein (FakA) that phosphorylates a fatty acid enveloped by a fatty acid binding protein (FakB). The structural basis for FakB function has been established, but little is known about FakA. Here, we used limited proteolysis to define three separate FakA domains: the amino terminal FakA_N, the central FakA_L, and the carboxy terminal FakA_C. The isolated domains lack kinase activity, but activity is restored when FakA_N and FakA_L are present individually or connected as FakA_NL. The X-ray structure of the monomeric FakA_N captures the product complex with ADP and two Mg 2+ ions bound at the nucleotide site. The FakA_L domain encodes the dimerization interface along with conserved catalytic residues Cys240, His282, and His284. AlphaFold analysis of FakA_L predicts the catalytic residues are spatially clustered and pointing away from the dimerization surface. Furthermore, the X-ray structure of FakA_C shows that it consists of two subdomains that are structurally related to FakB. Analytical ultracentrifugation demonstrates that FakA_C binds FakB, and site-directed mutagenesis confirms that a positively charged wedge on FakB meshes with a negatively charged groove on FakA_C. Finally, small angle X-ray scattering analysis is consistent with freely rotating FakA_N and FakA_C domains tethered by flexible linkers to FakA_L. These data reveal specific roles for the three independently folded FakA protein domains in substrate binding and catalysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗