Theses and Dissertations at Montana State University (MSU)
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Item New insights into radical initiation by radical S-adenosylmethionine enzymes and activation of [FeFe]-hydrogenase(Montana State University - Bozeman, College of Letters & Science, 2020) Impano, Stella; Chairperson, Graduate Committee: Joan B. Broderick; Hao Yang, Adrien Pagnier, Richard Jodts, Ryan Swimley, Eric M. Shepard, Sarah M. Hill, Christopher D. James, William E. Broderick, Brian M. Hoffman and Joan B. Broderick were co-authors of the article, 'Photolytic cleavage of S-adenosylmethionine' which is contained within this dissertation.; Eric M. Shepard, Hao Yang, Adrien Pagnier, Ryan Swimley, Emma Dolen, William E. Broderick, Brian M. Hoffman and Joan B. Broderick were co-authors of the article, 'Generation of an ethyl radical trapped in active sites of [FeFe]-hydrogenase maturase enzymes HydE AND HydG' which is contained within this dissertation.; Eric M. Shepard, Hao Yang, Jeremiah N. Betz, Adrien Pagnier, William E. Broderick, Brian M. Hoffman and Joan B. Broderick were co-authors of the article, 'EPR and ENDOR spectroscopic evidence of an ammonium binding site in HydE' which is contained within this dissertation.; Adrien Pagnier, Eric M. Shepard, William E. Broderick and Joan B. Broderick were co-authors of the article, 'Investigation into all the necessary components required for [FeFe]-hydrogenase H-cluster maturation' which is contained within this dissertation.; Dissertations contains two articles of which Stella Impano is not the main author.Radical S-adenosylmethionine (SAM) enzymes harbor a [4Fe-4S] cluster in their active sites that coordinates a catalytically relevant small molecule SAM. During catalysis the S-5'C bond of SAM is reductively cleaved to generate a 5'-deoxyadenosyl radical that subsequently abstracts an H atom from substrate, allowing functionally diverse reactions to be achieved. Trapping of the 5'-deoxyadenosyl radical intermediate during turnover had proven difficult likely due to the formation of omega intermediate resulting from the oxidative addition of the 5'-deoxyadenosyl radical to the unique iron of the cluster. Recently, our laboratory showed that this elusive 5'-deoxyadenosyl can be liberated, captured, and characterized, in the absence of substrate, via photoinduced electron transfer (ET)-mediated reductive cleavage of SAM. Further, photolysis of [4Fe-4S] +-SAM complexes in different radical SAM enzymes revealed that the regioselective bond cleavage of SAM is dependent on the active site environment where either a 5'-deoxyadenosyl or a *CH 3, depending on the enzyme. When Sadenosyl- ethionine is used in place of SAM in the [4Fe-4S] +-SAM complex of HydE or HydG an ethyl radical is trapped. In either case, annealing of the methyl and ethyl radicals yields corresponding omega-like species, omega M and omega E, respectively. Functionally, HydE and HydG work together with a third protein HydF, to synthesize the H-cluster of [FeFe]-hydrogenase enzymes. HydG lyses tyrosine to generate CO and CN - ligands of the diiron core of the H-cluster, while the role and substrate of HydE are yet to be elucidated; however, it is hypothesized that this enzyme is responsible for dithiomethylamine (DTMA) bridge assembly. Our hypothesis is that HydE uses ammonium as a co-substrate and we propose that this polyatomic ion condenses with two CH 2S- like species to assemble the DTMA. We demonstrate for the first time via EPR and ENDOR spectroscopic techniques that HydE harbors an ammonium binding site; this NH 4 + would be stored in the active site of HydE prior to DTMA synthesis. Additionally, through in vitro [FeFe]-hydrogenase assays, we investigate what component of the essential E. coli lysate is required for H-cluster assembly. Results from this work suggest that the Hyd maturases are not the only proteins needed for H-cluster biosynthesis.Item How biological catalysts activate oxygen to realize its full potential(Montana State University - Bozeman, College of Letters & Science, 2021) Ellis, Emerald Sue; Chairperson, Graduate Committee: Jennifer DuBois; Daniel J. Hinchen, Alissa Bleem, Lintao Bu, Bennett R. Streit, Quinlan V. Doolin, William E. Michener and Brandon C. Knott were authors and Sam J.B. Mallinson, Mark D. Allen, Melodie M. Machovina, Christopher W. Johnson, Gregg T. Beckham, John E. McGeehan, Jennifer L. DuBois were co-authors of the article, 'Engineering a biocatalyst for demethylation of lignin-derived aromatic aldehydes' in the journal 'Journal of the American Chemical Society Au' which is contained within this dissertation.; Luke MacHale, Robert K. Szilagyi and Jennifer L. DuBois were co-authors of the article, 'How chemical environment activates anthralin and molecular oxygen for direct reaction' in the journal 'Journal of organic chemistry' which is contained within this dissertation.; Dissertation contains an article of which Emerald Sue Ellis is not the main author.Dioxygen is a potent oxidant, inexpensive, and environmentally-friendly compared with most industrial oxidants, but intrinsic energy barriers to reaction limit its utility. Biological catalysts can activate O 2 by generating dangerous reactive oxygen species intermediates. The fundamental chemistry of two diverse O 2-utilizing enzyme systems were examined: GcoAB, a cytochrome P450 which catalyzes the O-demethylation of aromatic alcohols using heme to activate O 2, and NMO, an antibiotic biosynthesis monooxygenase which catalyzes cofactor-independent monooxygenation of an organic substrate. The enzyme active site environments and the reactions catalyzed therein were investigated with mutagenesis, X-ray crystallography, molecular dynamics simulations, fluorescence and UV/visible spectroscopy, cyclic voltammetry, electrode-based measurement of O 2 consumption, high-performance liquid chromatography, and simulations of homogenous solvation using quantum chemistry composite methods. The substrate range of GcoAB was expanded by rational design engineering to include two aromatic aldehydes commonly found in chemically-processed lignin. Only a single-point mutation was needed for GcoAB to catalyze demethylation of each new substrate. The reaction catalyzed by NMO can be called 'substrate-assisted' because the substrate mimics the role of the organic cofactor flavin in activating O 2. The physics of this reaction were probed using Marcus Theory, which relates the activation energy of the reaction to the free energy and the reorganization energy. By measuring the differences in the activation energy and free energy of the reaction within and without the enzyme, we found that the enzyme mainly acts on the reorganization energy term. The reaction was then examined in several homogenous solvents chosen based on their chemical similarity to individual amino acids. Homogenous solvation is much less computationally expensive to model than a protein active site, especially at higher levels of theory. By this approach, we discovered a plausible mechanism by which the chemical environment alone can boost the O 2-activating capacity of NMO's substrate--particularly by stabilizing the deprotonated anion which can transfer an electron to O 2 more easily than the neutral molecule. In summary, this work demonstrates that, while cofactors are responsible for activating O 2 in most oxidases, full appreciation of how an oxidase catalyzes reactions requires that neither the enzyme environment nor the substrate be ignored.Item Long term multispecies cover crops in semi-arid Montana: soil response and aboveground biomass(Montana State University - Bozeman, College of Agriculture, 2020) D'Agati, Kristen Mary; Chairperson, Graduate Committee: Catherine A. Zabinski; Perry Miller (co-chair); Perry R. Miller, Clain A. Jones and Catherine Zabinski were co-authors of the article, 'Soil biological effects of herbicide-terminated multi-species cover crop mixes, in semi-arid Montana' which is contained within this thesis.; Catherine Zabinski, Clain A. Jones and Perry R. Miller were co-authors of the article, 'Aboveground biomass quality and quantity of long-term multispecies cover crop mixes, in the semi-arid Montana' which is contained within this thesis.; Maryse Bourgault, Perry R. Miller, Clain A. Jones and Catherine Zabinski were co-authors of the article, 'Soil biological response to spraying, grazing, or haying of long-term multispecies cover crops in semi-arid Montana' which is contained within this thesis.Low and variable annual precipitation (250-350 mm) make management strategies that conserve soil moisture imperative for wheat producers in semi-arid Montana. A wheat-fallow rotation was historically the most common dryland cropping system in semi-arid Montana, due to its ability to conserve soil water; however, summer fallow has negative environmental impacts (Campbell et al., 1991). There is interest to incorporate cover crops into a rotation as a partial replacement for summer fallow to enhance soil quality. An eight-yr study explored the effect of cover crops on biological soil properties through aboveground biomass inputs of four plant functional groups: brassica (BC), fibrous root (FR), tap root (TR), and nitrogen fixers (NF) grown as two-species mixes, six-species mixes (three functional groups), a full eight-species mix, and two controls--chemical fallow and sole pea. Cover crops grew for about 60 days, were terminated with glyphosate, then soil samples were taken nine months after termination at wheat seeding. The only difference in biological parameters based on functional group was that mycorrhizal colonization in wheat was higher following FR than BC at one site. Potentially mineralizable nitrogen (PMN) was 1.6-1.7 times higher and microbial biomass was 1.4 times higher in soils from cover crop treatments relative to fallow at one of two sites. PMN was 1.2-1.3 times higher in soils from six-species mixes than two-species mixes at both sites, and six-species mixes produced 1.4 times more biomass at one site. Nitrogen fixers had the lowest C:N ratio of the functional groups at both sites, while FR had the highest at one site. In a second study of cover crop termination, cover crops were grown about 90 days and terminated with one of three strategies: chemically, grazing, or haying. Soils were sampled nine months after termination at the time of wheat seeding. Few enzyme differences and no PMN differences or meaningful patterns were discovered among termination strategies. Minimal differences in biological parameters, even when shoot biomass was removed, may mean grazing or haying could improve net revenue without detracting from soil health. In semi-arid annual systems, water limitations may be the main concern with growing cover crops.Item Biochemical and biophysical characterization of plastic degrading aromatic polyesterases(Montana State University - Bozeman, College of Letters & Science, 2019) Topuzlu, Ece; Chairperson, Graduate Committee: Valerie Copie; Brandon C. Knott and Mark D. Allen were authors and Japheth Gado, Harry P. Austin, Erika Erickson, Bryon S. Donohoe, Nicholas A. Rorrer, Fiona L. Kearns, Graham Dominick, Christopher W. Johnson, Valerie Copie, Christina M. Payne, H. Lee Woodcock, Gregg T. Beckham and John E. McGeehan were co-authors of the article, 'Structural and biochemical characterization of MHETASE' submitted to the journal 'Proceedings of the National Academy of Sciences of the United States of America' which is contained within this dissertation.As the world is producing more plastics than it can recycle, accumulation of manmade polymers in the environment is becoming one of the greatest global threats humanity is facing today. One of the major contributors to the plastics pollution problem is polyethylene terephthalate (PET), an aromatic polyester widely used in the packaging, beverage, garment and carpeting industries. As a response to the onslaught of plastics in the environment, fungi and bacteria are evolving metabolic pathways to convert plastics into useable energy sources. One of these organisms, a bacterium, Ideonella sakaiensis 201-F6, has recently been identified to convert PET into its monomers, terephthalic acid (TPA) and ethylene glycol (EG), and to use these compounds for energy and growth. I. sakaiensis' ability to convert PET is made possible by two enzymes, named PETase and MHETase. As a first step, PETase breaks down the insoluble substrate PET into a soluble major hydrolysis product - mono-(2- hydroxyethyl) terephthalate (MHET), which is then further hydrolyzed by MHETase into TPA and EG. Crystal structure of PETase, as well as some of its biochemical features, have been reported several times to date, but MHETase has remained largely uncharacterized. This work focuses on further discovery-driven biophysical and biochemical characterization of PETase, visualization of PETase activity on various polyester surfaces, as well as the structural and biochemical characterizations of the MHETase enzyme. We have found that several aspects of PETase-mediated substrate surface modification hydrolysis mechanisms differ depending on the specific mechanical and material characteristics of the substrate. We have also found that PETase is inhibited by BHET. Additionally, we have solved the crystal structure of MHETase. MHETase consists of an alpha/beta hydrolase domain, and a 'lid' domain, commonly seen in lipases. Molecular dynamics simulations revealed the mechanism of MHETase action. Through bioinformatics approaches, we have also identified mutants of interest for improved MHETase activity. Coincubation of MHETase with PETase affects PET turnover in a synergistic fashion. Taken together, this work provides additional insights into the mechanisms of action of the PETase and MHETase enzymes, which may open new avenue for bioremediation and removing plastics from the environment in a sustainable manner.Item Urease immobilization for advancing enzyme-induced calcium carbonate precipitation applications(Montana State University - Bozeman, College of Engineering, 2019) Frieling, Zachary James; Chairperson, Graduate Committee: Robin Gerlach and Adrienne Phillips (co-chair)Microbially induced calcium carbonate precipitation (MICP) is a widely studied field of research exploiting bacterial activity to form a calcium carbonate precipitate that has been used to modify porous media. MICP is an enzymatically driven process and uses the enzyme urease to change solution chemistry to favor calcium carbonate precipitation. An enzyme slurry can be used in lieu of microbial growth and can be applied in a similar fashion and is commonly referred to as enzymatically induced calcium carbonate precipitation (EICP). For some applications temperature can stunt microbial growth and EICP may be the preferred method. However, as the temperature increases further the urease enzyme is thermally inactivated inhibiting calcium carbonate precipitation. Thermal inactivation limits the potential use of EICP in higher temperature environments. To combat thermal inactivation, immobilization of the urease enzyme through entrapment in silica gel and adsorption on an internally porous ceramic proppant was evaluated, and the first order inactivation coefficient (kd) was determined for temperatures between 60°C and 90°C. It was found that immobilization of the urease enzyme drastically reduced the apparent k d when compared to the free, non-immobilized form. Column experiments were performed using the urease immobilized on the ceramic proppant at room temperature (~23°C) and at 60°C. It was found that the immobilized urease retained high activity for the duration of the experiments even when subjected to the elevated temperature condition. The immobilized form of the urease enzyme was indeed protected from thermal degradation. It also seemed that the immobilized form of the urease enzyme was shielded from inactivation from active calcium carbonate precipitation, as observed in previous EICP and MICP experiments, in which ureolytic activity decreased rapidly as calcium carbonate precipitated. As a result, the immobilized form of the urease enzyme showed promise for advancing EICP applications.Item Investigation of octopamine-glutamate dual transmission neurons(Montana State University - Bozeman, College of Letters & Science, 2020) McKinney, Hannah Margaret; Chairperson, Graduate Committee: Steven R. Stowers; Lewis Sherer, Jessica L. Williams, Sarah Certel and Steven R. Stowers were co-authors of the article, 'Characterization of drosophila mimic-converted octopamine receptor GAL4 lines' in the journal 'Journal of Comparative Neurology' which is contained within this dissertation.; Dissertation contains a paper of which Hannah Margaret McKinney is not the main author.Dual transmission, or the ability of a neuron to signal with more than one neurotransmitter, is now a well-established phenomenon in the field of neuroscience. However, many questions about this type of signaling process still remain with regards to its mechanisms and its impacts on neural circuitry and organism behavior. In particular, the mode of neurotransmitter release from synaptic vesicles can have significant profoundly affects elements on neural circuitry and, subsequently, on behaviors of an organism. In Drosophila melanogaster, a particular subset of neurons important for the behaviors of courtship and aggression signal with the neuromodulator octopamine and the excitatory neurotransmitter glutamate. Whether these two neurotransmitters are released simultaneously (co-release) or are housed for separate synaptic release (cotransmission) is unknown. The mechanism of release for these neurotransmitters in this population of neurons is investigated here through the development of synaptic vesicle visualization tools, synaptic vesicle isolation, and an examination of the expression of octopamine and glutamate receptors; I explored the hypothesis that receptor expression downstream of dual transmitting neurons will provide information about the co-release or co-transmission of octopamine and glutamate. Results from these experiments demonstrated release of octopamine and glutamate from the same synaptic site, with some variation, and a significant amount of presynaptic receptor expression. The results indicate these dual transmission neurons may release octopamine and glutamate at the same synapse for both post-synaptic signaling as well as pre-synaptic signal modulation.Item The reactive form of a C-S bond-cleaving CO 2-fixing flavoenzyme(Montana State University - Bozeman, College of Letters & Science, 2019) Mattice, Jenna Rose; Chairperson, Graduate Committee: Jennifer DuBois; Thesis includes a paper of which Jenna R. Mattice is not the main author.Atmospheric carbon dioxide (CO 2) is used as a carbon source for building biomass in plants and most engineered synthetic microbes. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme on earth, is used by these organisms to catalyze the first step in CO 2 fixation. 1,2 Microbial processes that also fix carbon dioxide or bicarbonate have more recently been discovered. My research focuses on a reaction catalyzed by 2-KPCC (NADPH:2-ketopropyl-coenzyme M oxidorectuase/ carboxylase), a bacterial enzyme that is part of the flavin and cysteine-disulfide containing oxidoreductase family (DSORs) which are best known for reducing metallic or disulfide substrates. 2-KPCC is unique because it breaks a comparatively strong C-S bond, leading to the generation of a reactive enolacetone intermediate which can directly attack and fix CO 2. 2-KPCC contains a phenylalanine in the place where most other DSOR members have a catalytically essential histidine. This research focuses on studying the unique reactive form of 2-KPCC in presence of an active site phenylalanine.Item Diffusion and diffusive exchange are sensitive to the structure of cartilage as measured by nuclear magnetic resonance(Montana State University - Bozeman, College of Engineering, 2017) Mailhiot, Sarah Elizabeth; Chairperson, Graduate Committee: Ronald K. June II; Nathan H. Williamson, Jennifer R. Brown, Joseph D. Seymour, Sarah L. Codd and Ronald K. June were co-authors of the article, 'T1-T2 correlation and biopolymer diffusion within human osteoarthritic cartilage measured with nuclear magnetic resonance' in the journal 'Applied magnetic resonance' which is contained within this thesis.; Sarah L. Codd, Jennifer R. Brown, Joseph D. Seymour and Ronald K. June were co-authors of the article, 'Pulsed gradient stimulated echo (PGSTE) NMR shows spatial dependence of fluid diffusion in human stage IV OA cartilage' submitted to the journal 'Magnetic resonance in medicine' which is contained within this thesis.; Fangrong Zong, James E. Maneval, Ronald K. June, Petrik Galvosas and Joseph D. Seymour were co-authors of the article, 'Quantifying NMR relaxation correlation and exchange in articular cartilage with time domain analysis' submitted to the journal 'Journal of magnetic resonance' which is contained within this thesis.; James E. Maneval, Ronald K. June and Joseph D. Seymour were co-authors of the article, 'Relaxation exchange in human OA cartilage impacts the observable T 2 relaxation rates' submitted to the journal 'Magnetic resonance in medicine' which is contained within this thesis.Osteoarthritis (OA) is the deterioration of the tissue on the surface of the articulating joints in mammals. OA is the progression loss of articular cartilage. OA affects 50% of people over age 65 and is the leading cause of workplace disability. There is no cure for OA and the state of the art treatment is joint replacement. One limitation for treating OA is the difficulty of diagnosing OA before tissue failure. Magnetic Resonance Imaging (MRI) is capable of detecting early pathologic changes to cartilage but challenges remain. The goal of this work is to evaluate how parameters, specifically relaxation and diffusion, used for creating imaging contrast in MRI are affected by disease in naturally occurring human osteoarthritis. Nuclear Magnetic Resonance (NMR) is utilized to measure the diffusion and magnetic relaxation in human OA cartilage samples. Diffusion Weighted Imaging (DWI) is a proposed imaging mechanism for diagnosing OA. The hypothesis is that fluid diffusion is faster in diseased tissue than in healthy tissue. We show that diffusion of fluid increases when cartilage is damaged by enzymes, such as during OA. We also show that the diffusion of fluid is donor specific in human OA cartilage. Diffusion of proteins in cartilage is also sensitive to enzyme degradation and donor as well as to the size and structure of the proteins in cartilage. These are complementary measures of the fluid and solid phase of cartilage. Relaxation weighted imaging is the most common way to image cartilage and is capable of measuring small structure changes due to OA. One limitation of this method is that reported relaxation rates vary between studies. We show that exchange, or motion of fluid, between the two sites of relaxation in cartilage alters the observed relaxation. Further, we show that the exchange rate is sensitive to donor and enzyme degradation. The results suggest that exchange rate is a sensitive measure of structure in cartilage and that relaxation should be cautiously interpreted when exchange occurs. Overall, this work shows that NMR and MRI are sensitive to the structure of cartilage and capable of detecting pathological damage to cartilage.Item Kinetics of thermally inactivated ureases and management of sand production through ureolysis-induced mineral precipitation(Montana State University - Bozeman, College of Engineering, 2018) Morasko, Vincent John; Chairperson, Graduate Committee: Robin Gerlach; Adrienne Phillips (co-chair)Biocement has the potential to seal subsurface hydraulic fractures, manipulate subsurface flow paths to enhance oil recovery, treat fractured cement, stabilize soil structures and minimize dust dispersal. Biocement can be formed using the urease enzyme from various sources (bacteria, plant, or fungi) to break down urea into carbonate, combining with calcium for use in engineering applications such as biocement production. Higher temperatures, pressures, and extreme pH conditions may be encountered as these engineering applications expand deeper into the subsurface. Temperatures beyond 1000 meters can exceed 80°C, potentially rapidly inactivating the enzyme. The first part of this study focused on monitoring urea hydrolysis catalyzed by jack bean urease at temperatures ranging from 20-80°C. An increasing rate of urease inactivation was observed with increasing temperatures and first-order models described the kinetics of urea hydrolysis and enzyme inactivation properly. The second part of this study focused on developing a technology to mitigate sand transport in oil and gas wells. This study addressed a method to cement sand in the subsurface so that it is not returned when oil or gas is extracted. As the sand leaves the formation, it can cause damage in the subsurface, leading to economic concerns, as well as reducing the lifespan of pumps, piping and other components on the well pad. A reactor system was developed to mimic a subsurface oil well that produces sand. Biocement production was promoted within the reactor, utilizing common sources of urease (Sporosarcina pasteurii and Canavalia ensiformis or jack bean meal). The resultant calcium carbonate/sand mass was subjected to elevated flowrates, simulating field conditions where sand is potentially fluidized and potentially transported into the wellbore. It was shown that biocement can reduce sand transport while allowing for higher flow rates than conditions without biocement. The findings from this study broaden the potential application range of biocementation technologies into higher temperature environments. Applying biocement specifically to sand mitigation may have significant environmental, economic, and safety implications within the natural resource industry.Item Evolution and function of flavin-based electron bifurcation(Montana State University - Bozeman, College of Letters & Science, 2018) Poudel, Saroj; Chairperson, Graduate Committee: Eric Boyd; John W. Peters (co-chair); Eric C. Dunham, Melody R. Lindsay, Maximiliano J. Amenabar, Elizabeth M. Fones, Daniel R. Colman and Eric S. Boyd were co-authors of the article, 'Origin and evolution of flavin-based electron bifurcating enzymes' in the journal 'Frontiers in microbiology' which is contained within this thesis.; Amaya M. Garcia Costas was an author and Anne-Frances Miller, Gerrit J. Schut, Rhesa N. Ledbetter, Kathryn R. Fixen, Lance C. Seefeldt, Michael W. W. Adams, Caroline S. Harwood, Eric S. Boyd and John W. Peters were co-authors of the article, 'Defining electron bifurcation in the electron transferring flavoprotein family' in the journal 'Journal of bacteriology' which is contained within this thesis.; Daniel R. Colman was an author and Kathryn R. Fixen, Rhesa N. Ledbetter, Yanning Zheng, Natasha Pence, Lance C. Seefeldt, John W. Peters, Caroline S. Harwood and Eric S. Boyd, were co-authors of the article, 'Electron transfer to nitrogenase in different genomic and metabolic backgrouns' in the journal 'Journal of bacteriology' which is contained within this thesis.Anaerobic microorganisms live in energy limited environments with low nutrient fluxes. Thus, selection has likely acted on these cells to innovate mechanisms that improve the efficiency of anaerobic energy metabolism. In 2008, the process of flavin-based electron bifurcation (FBEB) was discovered and has since been shown to be a critical process that allows anaerobic cells to overcome thermodynamic barriers and to improve metabolic efficiency. FBEB enzymes catalyze the coupling of exergonic and endergonic oxidation--reduction reactions with the same electron donor to circumvent thermodynamic barriers and minimize free energy loss. To date, a total of 12 FBEB enzymes have been discovered that share common features that include the presence of protein-bound flavin, the proposed site of bifurcation, and the electron carrier ferredoxin. Due to its recent discovery, a comprehensive description of the natural history of bifurcating enzymes is lacking. In this thesis, we report the taxonomic and ecological distribution, functional diversity, and evolutionary history of bifurcating enzyme homologs in available complete genomes and environmental metagenomes. Moreover, we investigated the functional and ecological constraints that led to the emergence of FBEB enzymes. Bioinformatics analyses revealed that FBEB enzyme homologs were primarily detected in the genomes of anaerobes, including those of sulfate-reducers, acetogens, fermenters, and methanogens. Phylogenetic analyses of these enzyme homologs suggest that they were not a property of the Last Universal Common Ancestor of Archaea and Bacteria indicating that they are a more recent evolutionary innovation. Consistent with the role of these enzymes in the energy metabolism of anaerobes, FBEB homologs were enriched in metagenomes from subsurface environments relative to those from surface environments. In fact, the earliest evolving homologs of most bifurcating enzymes were detected in subsurface environments, including fluids from subsurface rock fractures and hydrothermal systems. Together, these data highlight the central role that FBEB played and continued to play in the energy metabolism of anaerobic microbial cells inhabiting subsurface environments.