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At least 181 records · Page 10

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DOE OSTI2024

Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp 17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Identification of a Novel Cobamide Remodeling Enzyme in the Beneficial Human Gut Bacterium Akkermansia muciniphila

The beneficial human gut bacterium Akkermansia muciniphila provides metabolites to other members of the gut microbiota by breaking down host mucin, but most of its other metabolic functions have not been investigated. A. muciniphila strain Muc T is known to use cobamides, the vitamin B 12 family of cofactors with structural diversity in the lower ligand. However, A. muciniphila Muc T is unable to synthesize cobamides de novo, and the specific forms that can be used by A. muciniphila have not been examined. We found that the levels of growth of A. muciniphila Muc T were nearly identical with each of seven cobamides tested, in contrast to nearly all bacteria that had been studied previously. Unexpectedly, this promiscuity is due to cobamide remodeling—the removal and replacement of the lower ligand—despite the absence of the canonical remodeling enzyme CbiZ in A. muciniphila. We identified a novel enzyme, CbiR, that is capable of initiating the remodeling process by hydrolyzing the phosphoribosyl bond in the nucleotide loop of cobamides. CbiR does not share similarity with other cobamide remodeling enzymes or B12-binding domains and is instead a member of the apurinic/apyrimidinic (AP) endonuclease 2 enzyme superfamily. We speculate that CbiR enables bacteria to repurpose cobamides that they cannot otherwise use in order to grow under cobamide-requiring conditions; this function was confirmed by heterologous expression of cbiR in Escherichia coli. Homologs of CbiR are found in over 200 microbial taxa across 22 phyla, suggesting that many bacteria may use CbiR to gain access to the diverse cobamides present in their environment.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Signal sequences target enzymes and structural proteins to bacterial microcompartments and are critical for microcompartment formation

ABSTRACT Spatial organization of pathway enzymes has emerged as a promising tool to address several challenges in metabolic engineering, such as flux imbalances and off-target product formation. Bacterial microcompartments (MCPs) are a spatial organization strategy used natively by many bacteria to encapsulate metabolic pathways that produce toxic, volatile intermediates. Several recent studies have focused on engineering MCPs to encapsulate heterologous pathways of interest, but how this engineering affects MCP assembly and function is poorly understood. In this study, we investigated the role of signal sequences, short domains that target proteins to the MCP core, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterized two novel Pdu signal sequences on the structural proteins PduM and PduB, which constitute the first report of metabolosome signal sequences on structural proteins rather than enzymes. We then explored the role of enzymatic and structural Pdu signal sequences on MCP assembly by deleting their encoding sequences from the genome alone and in combination. Deleting enzymatic signal sequences decreased the MCP formation, but this defect could be recovered in some cases by overexpressing genes encoding the knocked-out signal sequence fused to a heterologous protein. By contrast, deleting structural signal sequences caused similar defects to knocking out the genes encoding the full-length PduM and PduB proteins. Our results contribute to a growing understanding of how MCPs form and function in bacteria and provide strategies to mitigate assembly disruption when encapsulating heterologous pathways in MCPs. IMPORTANCE Spatially organizing biosynthetic pathway enzymes is a promising strategy to increase pathway throughput and yield. Bacterial microcompartments (MCPs) are proteinaceous organelles that many bacteria natively use as a spatial organization strategy to encapsulate niche metabolic pathways, providing significant metabolic benefits. Encapsulating heterologous pathways of interest in MCPs could confer these benefits to industrially relevant pathways. Here, we investigate the role of signal sequences, short domains that target proteins for encapsulation in MCPs, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterize two novel signal sequences on structural proteins, constituting the first Pdu signal sequences found on structural proteins rather than enzymes, and perform knockout studies to compare the impacts of enzymatic and structural signal sequences on MCP assembly. Our results demonstrate that enzymatic and structural signal sequences play critical but distinct roles in Pdu MCP assembly and provide design rules for engineering MCPs while minimizing disruption to MCP assembly.

Johnson, Elizabeth R. (ORCID:0000000179236881)↗

DOE OSTI2024

Identification, characterization, and structure of a tRNA splicing enzyme RNA 5′-OH kinase from the pathogenic fungi Mucorales

Fungal Trl1 is an essential tRNA splicing enzyme composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that convert the 2′,3′-cyclic-PO 4 and 5′-OH ends of tRNA exons into the 3′-OH,2′-PO 4 and 5′-PO 4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trifunctional Trl1 enzymes are present in most human fungal pathogens and are untapped targets for antifungal drug discovery. Mucorales species, deemed high-priority human pathogens by WHO, elaborate a noncanonical tRNA splicing apparatus in which a stand-alone monofunctional RNA ligase enzyme joins 3′-OH,2′-PO 4 and 5′-PO 4 termini. Here we identify a stand-aloneMucor circinelloidespolynucleotide kinase (MciKIN) and affirm its biological activity in tRNA splicing by genetic complementation in yeast. Recombinant MciKIN catalyzes magnesium-dependent phosphorylation of 5′-OH RNA and DNA ends in vitro. MciKIN displays a strong preference for GTP as the phosphate donor in the kinase reaction, a trait shared with the stand-alone RNA kinase homologs from Mucorales speciesRhizopus azygosporus(RazKIN) andLichtheimia corymbifera(LcoKIN) and with the kinase domains of fungal Trl1 enzymes. We report a 1.65 Å crystal structure of RazKIN in complex with GDP•Mg 2+ that illuminates the basis for guanosine nucleotide specificity.

Biochemistry & Molecular Biology↗

DOE OSTI2022

Allosteric control of electron transfer in nitrogenase and nitrogenase-like enzymes (Final Report)

More than a third of all known proteins to date are either oxidoreductases or metalloproteins. The diversity of metal cofactors and redox centers in these proteins make them attractive candidates for engineering protein-based biocatalysts, biosensors, and biofuel cells, among other necessary tools for the bioeconomy. Many of these enzymes function as large multi-subunit complexes arranged as symmetric or asymmetric assemblies, and the purpose of this architecture is unclear. The goal of the project was to address this fundamental question by investigating the mechanism of action of two oxidoreductases: Nitrogenase and DPOR (Dark-Operative Protochlorophyllide Oxidoreductase, a nitrogenase-like oxidoreductase). Nitrogenase and DPOR play key roles in nitrogen fixation and photosynthesis, processes essential to sustaining life. Both proteins are arranged as two symmetrical halves with each half capable of substrate reduction. For more than half a century, substrate reduction by nitrogenase has been presumed to occur independently within the two identical halves. Funded by this DOE grant we have now shown that both nitrogenase and DPOR function asymmetrically. Electron transfer in one half suppresses activity in the other. Thus, we have demonstrated that the two halves do not function in an independent manner; rather, catalytic events in one-half allosterically suppress activity and electron transfer (ET) in the other. This alternating-site mechanism has significant implications on our current understanding of substrate reduction by these enzymes and offers a new mechanistic paradigm for the reactions they catalyze. Our work now provides a mechanistic map of the how the two symmetric catalytic halves work together to establish and maintain the sequential-site mechanism through multiple ET cycles to achieve substrate reduction. There is substantive need for bio-inspired tools that enable reduction of nitrogen and other substrates, as well as photosynthesis, for energy and food production. Understanding the workings of enzymes like nitrogenase and DPOR that mediate multi-electron redox chemistry is vital for designing such synthetic tools. The timing of our research coincides with the biotechnological breakthrough of light-driven artificial N2 reduction by nitrogenase, achieved using the native enzyme with both halves arranged in a mirror-like architecture as catalyst. Together with such discoveries, our research now reveal unknown facets of the reaction mechanism and hold promise for designing more effective catalysts.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Enzyme-assisted liquefaction for different fractions of corn stover pellets

With an increase in population, the world will depend on renewable sources to meet the increasing energy needs. The use of lignocellulosic biomass as a renewable source has been proven efficient for conversion to cellulosic ethanol and capable of contributing to thresholds for energy demand while reducing greenhouse gases by 90% when compared with fossil fuels. However, limitations in feeding and flow within biorefineries are encountered when system plugging occurs due to biomass compaction and a high yield stress for slurries formed during its processing. Furthermore, differences in the biomass source present a challenge to the continuity of operations and reliability in the process. This work explores enzyme-assisted liquefaction for solid loadings up to 30% as an alternative for the creation of slurry from two different types of biomass (pelleted corn stover and cobs) that were liquefied in a fed-batch process using commercial enzymes Celluclast 1.5L or Ctec-2 at 1FPU/g or 3 FPU/g of dry solids in a 10 mM sodium citrate buffer solution (pH 4.8). Pellets were fed into a 1 L stirred bioreactor according to a predefined fed-batch protocol during the first 5 hours until 30% solid loading was reached. After 6, 24 and 96 hours, samples were taken and characterized with respect to their composition, rheology, and water absorption. Successful slurry creation with dramatically reduced yield stress was achieved for corn stover for both enzymes assessed. Yield stresses of 178±7 Pa (3 FPU, Celluclast 1.5L) and 79±6 Pa (3 FPU, Ctec-2) were measured for corn stover at 24 hours, compared to 6,000 Pa for samples without enzyme. Yield stress was 155± 29 Pa (3FPU, Ctec-2) and 257 ± 72 Pa (1 FPU, Celluclast 1.5L) for corn cobs at 24 hours. A profile for 6, 24 and 96h of yield stress measurements and sugar conversion is presented.

Gutierrez, Diana↗

DOE OSTI2024

Development of Enzyme Production Host (CRADA Final Report)

This CRADA aims to assess the effectiveness of various enzyme production hosts in efficiently expressing and producing proprietary enzymes for plastic recycling applications. The research includes screening different microbial hosts—notably Bacillus subtilis, Pichia pastoris, and E. coli—to determine their suitability for high-yield enzyme production. Additionally, the project focuses on optimizing scalable fermentation processes tailored to these hosts, with the goal of enabling efficient enzyme production at commercially viable scales.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Development of Enzyme Production Host (CRADA Final Report)

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Sustainable production of plastic degrading enzymes

The 21st century faces the challenge of transitioning to a climate-neutral economy. This hurdle will need to be addressed in a myriad of approaches, including all societal spheres, as highlighted in several UN and EU reports, including new and accessed technologies. Among them, biotechnology can play a core role by exploiting its environmental benefits (e.g., substituting fossil-based products and expected greenhouse emission reductions). In some cases, biotech can even outperform fossil counterparts' features (e.g., the biodegradability of some bioplastics). Microalga is an attractive bioplastic feedstock in this scenario since it directly substitutes petroleum and captures CO2. However, albeit its environmental benefits, the microalgal process still needs to be tipped economically and the net-positive ecological impact improved to drive adoption. To that end, we envisioned the use of a green alga as a source of bioplastic feedstock and industrial enzymes, combining natural traits of microalgae with engineered properties in a resourceful process to petroleum-based products replacement. As proof of concept, we demonstrated that we could use C. reinhardtii, a green algae, to produce plastic-degrading enzymes. We assemble a vector to secrete PHL7, a recently described PETase, capable of degrading post-consumer PET plastic. We successfully engineered the cell line to secrete the plastic-degrading enzyme. We confirmed the secretion of the enzyme by observing halos in agar plates supplemented with plastic dispersions, enzymatic assays, zymograms, and protein sequencing of supernatant samples. This approach allows using green algae to replace petrochemical-derived plastic while producing the tool necessary to recycle existing plastics sustainably.

Dutra Molino, Joao Vitor↗

DOE OSTI2021

The Santorini Volcanic Complex as a Valuable Source of Enzymes for Bioenergy

Marine microbial communities are an untapped reservoir of genetic and metabolic diversity and a valuable source for the discovery of new natural products of biotechnological interest. The newly discovered hydrothermal vent field of Santorini volcanic complex located in the Aegean Sea is gaining increasing interest for potential biotechnological exploitation. The conditions in these environments, i.e., high temperatures, low pH values and high concentration of heavy metals, often resemble harsh industrial settings. Thus, these environments may serve as pools of enzymes of enhanced catalytic properties that may provide benefits to biotechnology. Here, we screened 11 metagenomic libraries previously constructed from microbial mat samples covering the seafloor and the polymetallic chimneys of Kolumbo volcano as well as mat samples from Santorini caldera, to mine, in silico, genes associated with bioenergy applications. We particularly focused on genes encoding biomass hydrolysis enzymes such as cellulases, hemicellulases and lignin-degrading enzymes. A total of 10,417 genes were found for three specific groups of enzymes—i.e., the endoglucanases, the three different beta-glucosidases BGL, bglX and bglB, and the alpha-galactosidases melA, and rafA. Overall, we concluded that the Santorini–Kolumbo volcanic ecosystems constitute a significant resource of novel genes with potential applications in bioenergy that deserve further investigation.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1972

Enzyme activity in terrestrial soil in relation to exploration of the Martian surface

An exploration was made of enzyme activities in soil, including abundance, persistence and localization of these activities. An attempt was made to develop procedures for the detection and assaying of enzymes in soils suitable for presumptive tests for life in planetary soils. A suitable extraction procedure for soil enzymes was developed and measurements were made of activities in extracts in order to study how urease is complexed in soil organic matter. Mathematical models were developed, based on enzyme action and microbial growth in soil, for rates of oxidation of nitrogen as nitrogen compounds are moved downward in soil by water flow. These biogeochemical models should be applicable to any percolating system, with suitable modification for special features, such as oxygen concetrations, and types of hydrodynamic flow.

Ardakani, M. S.↗

NASA NTRS1973

The effects of stress on the enzymes of peripheral leukocytes

Previous work showed an early response of rabbit and human leukocyte enzymes to the stress of bacterial infection. Since these represented a mixed population of leukocytes and since polymorphonuclear leukocytes (PMN) increased in these preparations, it was necessary to establish whether the observed increase in lactate dehydrenase (LDH) and protein was the result of an increase in any one particular cell type or in all cells. The need for the development of a simple reproducible method for the differential separation of peripheral leukocytes for the furtherance of our own studies was apparent. It was also becoming increasingly apparent that morphologically similar cells, such as small lymphocytes (L) and macrophages, were capable of different biological functions. A dextran gradient centrifugation method was developed which has provided an easily reproducible technique for separating L from PMN. During the course of this work, in which over 250 rabbits were examined, the pattern of daily leukocyte protein and enzyme variation became increasingly more apparent. This information could have some impact on future work with leukocyte enzymes, by our group and by other workers. The differences in normal protein and enzyme levels maintained by some individuals, and some inbred strains, were evaluated and reported separately. It has been shown that one type of leukocyte may react more to a given stress than other leukocytes.

Leise, E. M.↗

NASA NTRS1992

Effects of microgravity and tail suspension on enzymes of individual soleus and tibialis anterior fibers

Selected enzymes of energy metabolism were measured in random individual fibers of soleus and tibialis anterior (TA) muscles from rats exposed for 2 wk to spaceflight (F) aboard Cosmos 2044 or tail suspension (T) and from synchronous controls. Average size of soleus fibers (dry weight per unit length) was reduced 37 percent in F and T fibers; there was little change in Ta fibers. Enzyme changes were more pronounced in soleus than in TA fibers. Three enzymes characteristic of fast-twitch muscles, pyruvate kinase, glycerol-3-phosphate dehydrogenase, and 1-phosphofructokinase, were elevated in F and T soleus fibers, but changes in phosphofructokinase were not statistically significant. In TA fibers analyzed for hexokinase, malate dehydrogenase, phosphohexoisomerase, and pyruvate kinase, only hexokinase and malate dehydrogenase showed significant changes. Hexokinase incresed 83 percent in one of two T muscles. Enzyme data for TA fibers typed by myosin adenosinetriphosphatase were more informative: phosphofructokinase, phosphorylase, and glycerol-3-phosphate dehydrogenase were increased in type IIn fibers of either F or T muscles or both. Malate dehydrogenase was not changed in fibers of any type in either F or T muscle.

Chi, Maggie M.-Y.↗

NASA NTRS1973

Studies of a Halophilic NADH Dehydrogenase. 1: Purification and Properties of the Enzyme

An NADH dehydrogenase obtained from an extremely halophilic bacterium was purified 570-fold by a combination of gel filtration, chromatography on hydroxyapatite, and ion-exchange chromatography on QAE-Sephadex. The purified enzyme appeared to be FAD-linked and bad an apparent molecular weight of 64000. Even though enzyme activity was stimulated by NaCl, considerable activity (430 % of the maximum activity observed in the presence of 2.5 M NaCl) was observed in the absence of added NaCl. The enzyme was unstable when incubated in solutions of low ionic strength. The presence of NADH enhanced the stability of the enzyme.

Hochstein, Lawrence I.↗

NASA NTRS1976

Studies on the "Aerobic" Acetyl-Coenzyme A Synthetase of Saccharomyces Cerevisiae: Purification, Crystallization, and Physical Properties of the Enzyme

A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis. Sedimentation velocity runs revealed a single symmetric peak with an s(sub (20,w)) value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 +/- 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 +/- 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic Saccharomyces cerevisiae is composed of three subunits of identical or nearly identical size.

Satyanarayana, T.↗

NASA NTRS2002

Effects of Microgravity On Oxidative and Antioxidant Enzymes In Mouse Hindlimb Muscle

Gastrocnemius muscle of mice were analyzed in order to examine the effects of 12 days of microgravity on the oxidative enzyme climate synthase (CS) and the antioxidant enzyme superoxide dismutase (SOD). The female C57BL/6J mice utilized for this study were part of the Commercial Biomedical Testing Module (CBTM) payload that flew aboard STS-108. Mice were housed in Animal Enclosure Modules (AEMs) provided by NASA Ames. The flight (FLT) group and the ground control (CON) group each had 12 mice per group. The AEMs that held the CON group operated on a 48-hour delay from the FLT group and were located inside the Orbital Environmental Simulator (OES) at Kennedy Space Center. The temperature, CO2 and relative humidity inside the OES was regulated based on downlinked information from the shuttle middeck. Student T tests were used to compare groups and a p < 0.05 was used to determine statistical significance. Results indicated that CS levels for the FLT group were significantly lower than the CON group while the SOD levels were significantly higher. The CS FLT mean was 19% lower and the SOD FLT mean was 17% higher than the respective CON group means. Although these findings are among the first muscle enzyme values reported for mice from a shuttle mission, these results are similar to some results previously reported for rats exposed to microgravity or hindlimb suspension. The changes seen during the CBTM payload are reflective of the deconditioning that takes place with disuse of the hindlimbs and indicate that muscle enzyme changes induced by disuse deconditioning are similar in both rodent species.

Girten, B.↗

NASA NTRS2000

A DNA enzyme with N-glycosylase activity

In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

Non-NASA Center↗

DOE OSTI2021

Cytochrome P450 BM3 enzyme variants for preparation of cyclopropanes

The present invention provides methods for catalyzing the conversion of an olefin to any compound containing one or more cyclopropane functional groups using heme enzymes. In certain aspects, the present invention provides a method for producing a cyclopropanation product comprising providing an olefinic substrate, a diazo reagent, and a heme enzyme; and admixing the components in a reaction for a time sufficient to produce a cyclopropanation product. In other aspects, the present invention provides heme enzymes including variants and fragments thereof that are capable of carrying out in vivo and in vitro olefin cyclopropanation reactions. Expression vectors and host cells expressing the heme enzymes are also provided by the present invention.

Coelho, Pedro S.↗