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Results for “Multienzyme complexes”

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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Deconstruction by C. thermocellum —from microbe mediated to dynamic redistribution of cellulosomes

Clostridium thermocellum is one of the most efficient microorganisms for the deconstruction of cellulosic biomass. To achieve this high level of cellulolytic activity, C. thermocellum uses large multienzyme complexes known as cellulosomes to break down complex polysaccharides, notably cellulose, found in plant cell walls. The attachment of bacterial cells to the nearby substrate via the cellulosome has been hypothesized to be the reason for this high efficiency. The region lying between the cell and the substrate has shown great variation and dynamics that are affected by the growth stage of cells and the substrate used for growth. Here, we used both super-resolution imaging and machine-learning approaches to study the distribution of C. thermocellum cellulosomes at different stages of growth. We show that C. thermocellum initially retains its cellulosomes primarily on the cell surface but then relocates large cellulosome clusters to the interface with biomass, therefore depleting its cell surface of cellulosomes. These results indicate dynamic redistribution of cellulosomes during growth, with a functional shift toward substrate-associated degradation later during growth on biomass.

09 BIOMASS FUELS↗

Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

Ciszak, Ewa↗

Leveraging Super High Optical Resolution Microscopy to Probe the Interaction Zone Between Clostridium thermocellum and Biomass

This poster, part of the Pacific Northwest National Laboratory-sponsored Integration 2020: Visualizing the Proteome virtual conference, discusses super high optical resolution microscopy to probe the interaction zone between Clostritium thermocellum and biomass. Clostridium thermocellum is one of the most efficient microorganisms for the deconstruction of biomass. To achieve this high level of cellulolytic activity, C. thermocellum uses large multienzyme complexes known as cellulosomes to sugars in 4-5 break down polysaccharides found in plant cell walls. The attachment of bacterial switchgrass 70% cells to the nearby substrate via the cellulosome has been hypothesized to be the reason for this high efficiency. The region lying between the cell and the substrate has shown great variation and dynamics that are affected by the growth stage of cells and the substrate used for growth.

bacteria↗

Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module

Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. The bacillamide biosynthetic pathway is a common, three-protein system, with a decarboxylase that prepares an NRPS substrate, an NRPS, and an oxidase. Here, the pathway is reconstituted in vitro. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. We visualized co-complexes of the oxidase bound to the elongation module of the NRPS using X-ray crystallography and cryo-EM. The three active sites (for adenylation, condensation/cyclization, and oxidation) form an elegant arc to facilitate substrate delivery. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS.

59 BASIC BIOLOGICAL SCIENCES↗

Unnatural biosynthesis by an engineered microorganism with heterologously expressed natural enzymes and an artificial metalloenzyme

Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here, in this work, we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium–porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Cryo-EM structures of engineered active bc 1- cbb 3 type CIII 2 CIV super-complexes and electronic communication between the complexes

Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc 1 (Complex III, CIII 2 ), and may have specific cbb 3 -type cyt c oxidases (Complex IV, CIV) instead of the canonical aa 3 -type CIV. Electron transfer between these complexes is mediated by soluble ( c 2 ) and membrane-anchored ( c y ) cyts. Here, we report the structure of an engineered bc 1 - cbb 3 type SC (CIII 2 CIV, 5.2Å resolution) and three conformers of native CIII 2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt c y and the assembly factor CcoH. The cyt c y is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c 2 . The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria.

42 ENGINEERING↗

A metal ion orients SARS-CoV-2 mRNA to ensure accurate 2'- O methylation of its first nucleotide

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

60 APPLIED LIFE SCIENCES↗

Insight into the autoproteolysis mechanism of the RsgI9 anti‐σ factor from Clostridium thermocellum

Abstract Clostridium thermocellum is a potential microbial platform to convert abundant plant biomass to biofuels and other renewable chemicals. It efficiently degrades lignocellulosic biomass using a surface displayed cellulosome, a megadalton sized multienzyme containing complex. The enzymatic composition and architecture of the cellulosome is controlled by several transmembrane biomass‐sensing RsgI‐type anti‐σ factors. Recent studies suggest that these factors transduce signals from the cell surface via a conserved RsgI extracellular (CRE) domain (also called a periplasmic domain) that undergoes autoproteolysis through an incompletely understood mechanism. Here we report the structure of the autoproteolyzed CRE domain from the C. thermocellum RsgI9 anti‐σ factor, revealing that the cleaved fragments forming this domain associate to form a stable α/β/α sandwich fold. Based on AlphaFold2 modeling, molecular dynamics simulations, and tandem mass spectrometry, we propose that a conserved Asn‐Pro bond in RsgI9 autoproteolyzes via a succinimide intermediate whose formation is promoted by a conserved hydrogen bond network holding the scissile peptide bond in a strained conformation. As other RsgI anti‐σ factors share sequence homology to RsgI9, they likely autoproteolyze through a similar mechanism.

Takayesu, Allen↗

Structural insight on assembly-line catalysis in terpene biosynthesis

Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Structural and functional insights into δ-poly-L-ornithine polymer biosynthesis from Acinetobacter baumannii

Cationic homo-polyamino acid (CHPA) peptides containing isopeptide bonds of diamino acids have been identified from Actinomycetes strains. However, none has been reported from other bacteria. Here, we report a δ-poly-L-ornithine synthetase from Acinetobacter baumannii, which we name PosA. Surprisingly, structural analysis of the adenylation domain and biochemical assay shows L-ornithine as the substrate for PosA. The product from the enzymatic reaction was purified and identified as poly-L-ornithine composed of 7-12 amino acid units. Chemical labeling of the polymer confirmed the isopeptide linkage of δ-poly-L-ornithine. We examine the biological activity of chemically synthesized 12-mer δ-poly-L-ornithine, illustrating that the polymer may act as an anti-fungal agent. Structures of the isolated adenylation domain from PosA are presented with several diamino acids and biochemical assays identify important substrate binding residues. Structurally-guided genome-mining led to the identification of homologs with different substrate binding residues that could activate additional substrates. A homolog from Bdellovibrionales sp. shows modest activity with L-arginine but not with any diamino acids observed to be substrates for previously examined CHPA synthetases. Our study indicates the possibility that additional CHPAs may be produced by various microbes, supporting the further exploration of uncharacterized natural products.

59 BASIC BIOLOGICAL SCIENCES↗

Cellulosome Localization Patterns Vary across Life Stages of Anaerobic Fungi

Anaerobic fungi ( Neocallimastigomycota ) isolated from the guts of herbivores excel at degrading ingested plant matter, making them attractive potential platform organisms for converting waste biomass into valuable products, such as chemicals and fuels. Major contributors to their biomass-hydrolyzing power are the multienzyme cellulosome complexes that anaerobic fungi produce, but knowledge gaps in how cellulosome production is controlled by the cellular life cycle and how cells spatially deploy cellulosomes complicate the use of anaerobic fungi and their cellulosomes in industrial bioprocesses.

59 BASIC BIOLOGICAL SCIENCES↗

Self-Assembling Cell-Free Systems for Scalable Bioconversion

This project focused on developing cell-free systems to directly express multi-enzyme catalysts and perform CO2 bioconversions for industrial chemical production. The use of cell-free expression (CFE) systems derived from bacterial lysates is emerging as a promising approach for biomanufacturing. CFEs are genetically programmable, permit the expression of toxic enzymes, and allow for rapid prototyping of metabolic pathways. Research Contributions: 1. Understanding the Area Investigated: This research advances the understanding of cell-free systems by demonstrating their capability to perform complex multi-enzyme reactions. By directly expressing multi-gene systems, CFEs avoid the high costs and inefficiencies associated with producing and purifying enzymes for multi-step pathways. 2. Technical Effectiveness and Economic Feasibility: The project successfully engineered a CFE-based multienzyme biocatalyst for the de novo synthesis of serine and glycine from CO2 equivalents (formate and bicarbonate) and ammonia. This method achieved a 30% conversion rate of formate into these industrially important amino acids. Additionally, an 8-gene CFE biocatalyst was developed to produce malate, conserving 43% of carbon that would otherwise be lost as CO2. This approach has the potential to reach higher carbon efficiency than microbial production. 3. Public Benefit: The cell-free production of chemicals like serine, glycine, and malate using electrochemically generated formate could significantly reduce CO2 emissions. For example, satisfying the global malate market with this method could avoid approximately 400,000 tons of CO2 emissions annually. This work demonstrates the potential of CFE systems to produce platform chemicals, contributing to environmental sustainability and reducing reliance on petrochemicals. Future Prospects: The CFE-based biocatalyst process could be extended to produce a variety of chemicals, including other industrial di-acids, aromatics, terpenes, alcohols, and polymers. This project showcases the capabilities of cell-free expression systems for prototyping carbon-conserving pathways and sustainably bioproducing platform chemicals, marking a significant step towards economically-viable industrial processes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Auxin-activated NADH oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties

The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14-17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.

Non-NASA Center↗

NADH oxidase activity (NOX) and enlargement of HeLa cells oscillate with two different temperature-compensated period lengths of 22 and 24 minutes corresponding to different NOX forms

NOX proteins are cell surface-associated and growth-related hydroquinone (NADH) oxidases with protein disulfide-thiol interchange activity. A defining characteristic of NOX proteins is that the two enzymatic activities alternate to generate a regular period length of about 24 min. HeLa cells exhibit at least two forms of NOX. One is tumor-associated (tNOX) and is inhibited by putative quinone site inhibitors (e.g., capsaicin or the antitumor sulfonylurea, LY181984). Another is constitutive (CNOX) and refractory to inhibition. The periodic alternation of activities and drug sensitivity of the NADH oxidase activity observed with intact HeLa cells was retained in isolated plasma membranes and with the solubilized and partially purified enzyme. At least two activities were present. One had a period length of 24 min and the other had a period length of 22 min. The lengths of both the 22 and the 24 min periods were temperature compensated (approximately the same when measured at 17, 27 or 37 degrees C) whereas the rate of NADH oxidation approximately doubled with each 10 degrees C rise in temperature. The rate of increase in cell area of HeLa cells when measured by video-enhanced light microscopy also exhibited a complex period of oscillations reflective of both 22 and 24 min period lengths. The findings demonstrate the presence of a novel oscillating NOX activity at the surface of cancer cells with a period length of 22 min in addition to the constitutive NOX of non-cancer cells and tissues with a period length of 24 min.

Non-NASA Center↗

Cell enlargement of plant tissue explants oscillates with a temperature-compensated period of ca. 24 min

Rate of plant cell enlargement, measured at intervals of 3 min using a sensitive linear transducer, oscillates with a minimum period of about 24 min that parallels the 24-min periodicity observed with the oxidation of NADH by the external plasma membrane NADH oxidase and of single cells measured previously by video-enhanced light microscopy. Also exhibiting 24-min oscillations is the steady-state rate of cell enlargement induced by the addition of the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges. The length of the 24-min period is temperature compensated and remains constant at 24 min when measured at 15, 25 or 35 degrees C, despite the fact that the rate of cell enlargement approximately doubles for each 10 degree C rise over this same range of temperatures.

Non-NASA Center↗

The plasma membrane-associated NADH oxidase of spinach leaves responds to blue light

The plasma membrane-associated NADH oxidase (NOX) of spinach leaf disks is characterized by oscillations in activity with a regular period length of ca. 24 min. Within a single population of plants exposed to light at the same time, NOX activities of all plants function synchronously. Exposure of plants transferred from darkness to blue light (495 nm, 2 min, 50 micromoles m-2 s-1) resulted in a complex response pattern but with a new maximum in the rate of NOX activity 36 (24+12) min after illumination and then with maxima in the rate of NOX activity every 24 min thereafter. Transient maxima in NOX activity were observed as well after 9.3 + /- 1.4 and 20.7 +/- 2.1 min. The blue light response differed from the response to red (650 nm, 10 min, 50 micromoles m-2 s-1) or white light where activity maxima were initiated 12 min after the light exposure followed by maxima every 24 min thereafter. Green or yellow light was ineffective. The light response was independent of the time in the 24-min NOX cycle when the light was given. The net effects of blue and red light were ultimately the same with a new maximum in the rate of NOX activity at 12+24=36 min (and every 24 min thereafter), but the mechanisms appear to be distinct.

NASA Discipline Cell Biology↗

A monoclonal antibody that distinguishes latent and active forms of the proteasome (multicatalytic proteinase complex)

Monoclonal antibodies (mAbs) were generated to proteasome purified from human erythrocytes. Five of six proteasome-specific mAbs reacted with three subunits in the molecular mass range of 25-28 kDa, indicating a common epitope. The other mAb (AP5C10) exhibited a more restricted reactivity, recognizing a 32-kDa subunit of the proteasome purified in its latent state. However, when the proteasome is isolated in its active state, AP5C10 reacts with a 28-kDa subunit, evidence for processing of the proteasome subunits during purification. Purified proteasome preparations which exhibited partial latency have both AP5C10 reactive subunits. Although the 32-kDa subunit appears required for latency, loss of this component and generation of the 28-kDa component are not obligatory for activation. The 32- and 28-kDa subunits can each be further resolved into three components by isoelectric focusing. The apparent loss of 4 kDa during the conversion of the 32- to 28-kDa subunit is accompanied by a shift to a more basic pI for each polypeptide. Western blots of the early steps of proteasome purification reveal an AP5C10-reactive protein at 41 kDa. This protein was separated from proteasomes by sizing chromatography and may represent a pool of precursor subunits. Since the 32-kDa subunit appears necessary for latency, it is speculated to play a regulatory role in ATP-dependent proteolytic activity.

NASA Discipline Musculoskeletal↗