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At least 73 records · Page 4

Crystal Ball: A Naturalist Perspective of Microbiology: Examples from Methanogenic Archaea

Storytelling has been the primary means of knowledge transfer over human history. The effectiveness and reach of stories are improved when the message is appropriate for the target audience. Oftentimes, the stories that are most well received and recounted are those that have a clear purpose and that are told from a variety of perspectives that touch on the varied interests of the target audience. Whether scientists realize or not, they are accustomed to telling stories of their own scientific discoveries through the preparation of manuscripts, presentations, and lectures. Perhaps less frequently, scientists prepare review articles or book chapters that summarize a body of knowledge on a given subject matter, meant to be more holistic recounts of a body of literature. Yet, by necessity, such summaries are often still narrow in their scope and are told from the perspective of a particular discipline. In other words, interdisciplinary reviews or book chapters tend to be the rarity rather than the norm. Naturalists are scientists that tell stories of organisms and their natural environment within a historical context and from an interdisciplinary perspective. Such stories interweave observations of biological subjects from disparate disciplines into a cohesive story. To this end, the success of naturalists in recounting compelling natural history stories is enhanced when they allow themselves to become vulnerable by knowingly entering a literature base full of unfamiliar ways of observing and describing such subjects. When successful, this approach can broaden the scope of the story, making it of interest to a larger audience and attracting further recounting of that story (e.g., citations). To this end, when we look into our crystal ball, we see a need to tell more holistic stories of microorganisms and their functions from an interdisciplinary, naturalist perspective that combines what is known of their contemporary ecology, physiology, and biochemistry with their evolutionary history thereby placing them within a historical Earth context. With this conviction in mind, we take the opportunity to look back on the natural history of methanogens to identify some of their most profound biochemical innovations and their impact on the trajectory of life and the planet. Our approach was to integrate knowledge from the fields of biochemistry, physiology, ecology, evolution, geochemistry, and atmospheric sciences. We start with the origin of the first autotrophic cells and how this set Earth on a path towards biomass expansion and functional diversification. Next, we discuss the importance of iron and sulfur in methanogen metabolism, and how their innovative ways of accessing these elements allowed for expanded use of iron-sulfur clusters ([Fe-S]) as nanoscale catalysts that power(ed) Earth’s biochemical cycles. We next discuss the origin and evolution of [NiFe]-hydrogenase and how this versatile enzyme chassis was co-opted by a variety of anaerobes and aerobes as the foundation for respiratory complexes capable of sustaining higher forms of life, including humans. Lastly, we discuss the evolution of the enzyme molybdenum nitrogenase, and how this allowed the biosphere to overcome fixed nitrogen limitation by tapping the seemingly endless reserve of atmospheric dinitrogen (N 2 ). In telling these stories, we aimed to generate template(s) to motivate future interdisciplinary natural history studies focused on other organisms and processes. Further, we hope to stimulate scientific discourse and provide impetus to become vulnerable and peruse literature of relevance to understanding the causes and consequences of the evolution of your favorite microorganism or biochemical process.

59 BASIC BIOLOGICAL SCIENCES↗

Structural adaptation of oxygen tolerance in 4-hydroxybutyrl-CoA dehydratase, a key enzyme of archaeal carbon fixation

Abstract Autotrophic microorganisms that convert inorganic carbon into organic matter were key players in the evolution of life on Earth. As the early atmosphere became oxygenated, microorganisms needed to develop mechanisms for oxygen protection, especially those relying on enzymes containing oxygen-sensitive metal clusters (e.g., Fe-S). Here we investigated how 4-hydroxybutyryl-CoA dehydratase (4HBD) - the key enzyme of the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle for CO 2 -fixation - adapted as conditions shifted from anoxic to oxic. 4HBD is found in both anaerobic bacteria and aerobic ammonia-oxidizing archaea (AOA). The oxygen-sensitive bacterial 4HBD and oxygen-tolerant archaeal 4HBD share 59 % amino acid identity. To examine the structural basis of oxygen tolerance in archaeal 4HBD, we determined the atomic resolution structure of the enzyme. Two tunnels providing access to the canonical [4Fe-4S] cluster in oxygen-sensitive bacterial 4HBD were closed with four conserved mutations found in all aerobic AOA and other archaea. Further biochemical experiments and molecular dynamics simulations support our findings that restricting access to the active site is the key to oxygen tolerance, explaining how active site evolution drove a major evolutionary transition. Significance statement Autotrophy (primary production) was the first life strategy on Earth. Before photosynthesis (using solar energy to fix carbon dioxide), life relied on chemical reactions for energy. These chemosynthetic reactions are present in all domains of life, including archaea possessing the most energy-efficient carbon fixation pathway - the 3-hydroxypropionate/4-hydroxybutyrate cycle. This efficiency results from enzyme modifications, including enhanced enzyme stability and catalysis of multiple reactions. We reveal the first structure of aerobic 4-hydroxybutyryl-CoA dehydratase (4HBD) from ammonia-oxidizing archaea. These archaea are among the most abundant organisms on the planet, and their 4HBD active site evolved oxygen tolerance to support aerobic metabolism. This modification can provide further insight into enzyme evolution on early earth, as photosynthesis developed and began oxygenating the atmosphere.

DeMirci, Hasan (ORCID:0000000291355397)↗

Using enzymes to tame nitrogen-centred radicals for enantioselective hydroamination

The formation of C–N bonds—of great importance to the pharmaceutical industry—can be facilitated enzymatically using nucleophilic and nitrene transfer mechanisms. However, neither natural nor engineered enzymes are known to generate and control nitrogen-centred radicals, which serve as valuable species for C–N bond formation. Here we use flavin-dependent ‘ene’-reductases with an exogenous photoredox catalyst to selectively generate amidyl radicals within the protein active site. These enzymes are engineered through directed evolution to catalyse 5-exo, 6-endo, 7-endo, 8-endo, and intermolecular hydroamination reactions with high levels of enantioselectivity. Mechanistic studies suggest that radical initiation occurs via an enzyme-gated mechanism, where the protein thermodynamically activates the substrate for reduction by the photocatalyst. Molecular dynamics studies indicate that the enzymes bind substrates using non-canonical binding interactions, which may serve as a handle to further manipulate reactivity. Furthermore, this approach demonstrates the versatility of these enzymes for controlling the reactivity of high-energy radical intermediates and highlights the opportunity for synergistic catalyst strategies to unlock previously inaccessible enzymatic functions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Opportunities and Challenges for Machine Learning-Assisted Enzyme Engineering

Enzymes can be engineered at the level of their amino acid sequences to optimize key properties such as expression, stability, substrate range, and catalytic efficiency or even to unlock new catalytic activities not found in nature. Because the search space of possible proteins is vast, enzyme engineering usually involves discovering an enzyme starting point that has some level of the desired activity followed by directed evolution to improve its “fitness” for a desired application. Recently, machine learning (ML) has emerged as a powerful tool to complement this empirical process. ML models can contribute to (1) starting point discovery by functional annotation of known protein sequences or generating novel protein sequences with desired functions and (2) navigating protein fitness landscapes for fitness optimization by learning mappings between protein sequences and their associated fitness values. In this Outlook, we explain how ML complements enzyme engineering and discuss its future potential to unlock improved engineering outcomes.

60 APPLIED LIFE SCIENCES↗

Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer’s medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC 50 = 20 μM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4’-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations.

60 APPLIED LIFE SCIENCES↗

Enzymatic Nitrogen Incorporation Using Hydroxylamine

Hydroxylamine-derived reagents have enabled versatile nitrene transfer reactions for introducing nitrogen-containing functionalities in small-molecule catalysis as well as biocatalysis. These reagents, however, result in poor atom economy and stoichiometric organic waste. Activating hydroxylamine (NH 2 OH) for nitrene transfer offers a low-cost and sustainable route to amine synthesis, since water is the sole byproduct. Despite its presence in nature, however, hydroxylamine is not known to be used for enzymatic nitrogen incorporation in biosynthesis. Here, we report an engineered heme enzyme that can utilize hydroxylammonium chloride, an inexpensive commodity chemical, for nitrene transfer. Directed evolution of Pyrobaculum arsenaticum protoglobin generated efficient enzymes for benzylic C–H primary amination and styrene aminohydroxylation. Mechanistic studies supported a stepwise radical pathway involving rate-limiting hydrogen atom transfer. Furthermore, this unprecedented activity is a useful addition to the ‘nitrene transferase’ repertoire and hints at possible future discovery of natural enzymes that use hydroxylamine for amination chemistry.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

A molecular description of the evolution of resistance

BACKGROUND: In vitro evolution has been used to obtain nucleic acid molecules with interesting functional properties. The evolution process usually is carried out in a stepwise manner, involving successive rounds of selection, amplification and mutation. Recently, a continuous in vitro evolution system was devised for RNAs that catalyze the ligation of oligonucleotide substrates, allowing the evolution of catalytic function to be studied in real time. RESULTS: Continuous in vitro evolution of an RNA ligase ribozyme was carried out in the presence of a DNA enzyme that was capable of cleaving, and thereby inactivating, the ribozyme. The DNA concentration was increased steadily over 33.5 hours of evolution, reaching a final concentration that would have been sufficient to inactivate the starting population in one second. The evolved population of ribozymes developed resistance to the DNA enzyme, reducing their vulnerability to cleavage by 2000-fold but retaining their own catalytic function. Based on sequencing and kinetic analysis of the ribozymes, two mechanisms are proposed for this resistance. One involves three nucleotide substitutions, together with two compensatory mutations, that alter the site at which the DNA enzyme binds the ribozyme. The other involves enhancement of the ribozyme's ability to bind its own substrate in a way that protects it from cleavage by the DNA enzyme. CONCLUSIONS: The ability to direct the evolution of an enzyme's biochemical properties in response to the behavior of another macromolecule provides insight into the evolution of resistance and may be useful in developing enzymes with novel or enhanced function.

Non-NASA Center↗

Adaptive_Evolution_2026

Thermoanaerobacterium saccharolyticum, an anaerobic and thermophilic bacterium capable of metabolizing sugar monomers and soluble oligomers into ethanol, has been proposed for use in consolidated bioprocessing in coculture with compatible cellulolytic bacteria such asClostridium thermocellum. Although the mixed acid fermentation of both of these strains has been engineered to produce ethanol as the major fermentation product, the maximum titer produced thus far byC. thermocellum, about 3% (w/v) ethanol, is half that produced byT. saccharolyticum. There is thus motivation to understand the mechanistic basis of the robust ethanol pathway inT. saccharolyticumso that key features can be recapitulated inC. thermocellumand other organisms.Previously, we characterized theindividualrole of the main genes responsible for electron transfer in the ethanol production ofT. saccharolyticum. However, the consequences of thecombinedloss of function of all these genes have not been investigated, nor has the way in which fermentative metabolism adapts to such constraints. In this work, we combined knockouts of ferredoxin nicotinamide oxidoreductase (fnor) genes (nfnAandnfnB) and hydrogenase genes (hydAandhfsD) and studied their effects on fermentation and grow. We showed that these genetic modifications together impair growth and decrease electron transfer from reduced ferredoxin, thereby redirecting flux from the pyruvate ferredoxin oxidoreductase enzyme to the pyruvate formate lyase enzyme. We also performed adaptive evolution of these mutants to rescue their growth, and most notably, we observed a single nucleotide variation in the alcohol dehydrogenaseadhAgene. Through molecular dynamics simulations and enzymatic assays, we determined that this point mutation causes a structural change that impairs the AdhA specificity for the NADPH cofactor and increases NADH-linked activity to restore redox balance. These findings consolidate our understanding of the functioning of electron transfer pathways in this organism.

alcohol dehydrogenase↗

Cross-Kingdom Comparative Genomics of Aromatic Catabolic Pathways in Fungi and Bacteria

The overall goal of this project is to test the hypothesis that white-rot fungi can simultaneously depolymerize lignin extracellularly and catabolize depolymerization products intracellularly as carbon and energy sources. The results from this project will lead to improved understanding of lignin utilization by white-rot fungi, and enable identification of promising fungal strains for lignin catabolism and valorization. As part of this effort, we have conducted a comparative genomic study to investigate a diversity of aromatic catabolic pathways in the fungal and bacterial kingdoms. Furthermore, we have combined genomic and phylogenetic approaches to decipher the evolution of certain aromatic catabolic enzymes and discover new classes of enzymes, which represents a foundation for future biochemical and molecular genetic studies.

aromatic compounds↗

Rewinding evolution in planta: A Rubisco-null platform validates high-performance ancestral enzymes

Improving the photosynthetic enzyme Rubisco is a key target for enhancing C3 crop productivity, but progress has been hampered by the difficulty of evaluating engineered variants in planta without interference from the native enzyme. Here, we report the creation of a Rubisco-null Nicotiana tabacum platform by using CRISPR-Cas9 to knock out all 11 nuclear-encoded small subunit (rbcS) genes. Knockout was achieved in a line expressing cyanobacterial Rubisco from the plastid genome, allowing the recovery of viable plants. We then developed a chloroplast expression system for coexpressing both large and small subunits from the plastid genome. We expressed two resurrected ancestral Rubiscos from the Solanaceae family. The resulting transgenic plants were phenotypically normal and accumulated Rubisco to wild-type levels. Importantly, kinetic analyses of the purified ancestral enzymes revealed they possessed a 16 to 20% higher catalytic efficiency (k cat,air /K c,air ) under ambient conditions, driven by a significantly faster turnover rate (k cat,air ). We have demonstrated that our system allows robust in vivo assessment of novel Rubiscos and that ancestral reconstruction is a powerful strategy for identifying superior enzymes to improve photosynthesis in C3 crops.

59 BASIC BIOLOGICAL SCIENCES↗

High Throughput expression and characterization of laccases in Saccharomyces cerevisiae

Laccases are oxidative enzymes containing 4 conserved copper heteroatoms. Laccases catalyze cleavage of bonds in lignin using radical chemistry, yet their exact specificity for bonds (such as the β-O-4 or C-C) in lignin remains unknown and may vary with the diversity of laccases across fungi, plants and bacteria. Bond specificity may perhaps even vary for the same enzyme across different reaction conditions. Determining these differences has been difficult due to the fact that heterologous expression of soluble, active laccases has proven difficult. Here we describe the successful heterologous expression of functional laccases in two strains of Saccharomyces cerevisiae, including one we genetically modified with CRISPR. We phylogenically map the enzymes that we successfully expressed, compared to those that did not express. We also describe differences protein sequence differences and pH and temperature profiles and their ability to functionally express, leading to a potential future screening platform for directed evolution of laccases and other ligninolytic enzymes such as peroxidases.

59 BASIC BIOLOGICAL SCIENCES↗

Promising approaches for the assembly of the catalytically active, recombinant Desulfomicrobium baculatum hydrogenase with substitutions at the active site

Background: Hydrogenases (H2ases) are metalloenzymes capable of the reversible conversion of protons and electrons to molecular hydrogen. Exploiting the unique enzymatic activity of H2ases can lead to advancements in the process of biohydrogen evolution and green energy production. Results: Here we created of a functional, optimized operon for rapid and robust production of recombinant [NiFe] Desulfomicrobium baculatum hydrogenase (Dmb H2ase). The conversion of the [NiFeSe] Dmb H2ase to [NiFe] type was performed on genetic level by site-directed mutagenesis. The native dmb operon includes two structural H2ase genes, coding for large and small subunits, and an additional gene, encoding a specific maturase (protease) that is essential for the proper maturation of the enzyme. Dmb, like all H2ases, needs intricate bio-production machinery to incorporate its crucial inorganic ligands and cofactors. Strictly anaerobic, sulfate reducer D. baculatum bacteria are distinct, in terms of their biology, from E. coli. Thus, we introduced a series of alterations within the native dmb genes. As a result, more than 100 elements, further compiled into 32 operon variants, were constructed. The initial requirement for a specific maturase was omitted by the artificial truncation of the large Dmb subunit. The assembly of the produced H2ase subunit variants was investigated both, in vitro and in vivo. This approach resulted in 4 recombinant [NiFe] Dmb enzyme variants, capable of H 2 evolution. The aim of this study was to overcome the gene expression, protein biosynthesis, maturation and ligand loading bottlenecks for the easy, fast, and cost-effective delivery of recombinant [NiFe] H2ase, using a commonly available E. coli strains. Conclusion: The optimized genetic constructs together with the developed growth and purification procedures appear to be a promising platform for further studies toward fully-active and O 2 tolerant, recombinant [NiFeSe] Dmb H2ase, resembling the native Dmb enzyme. It could likely be achieved by selective cysteine to selenocysteine substitution within the active site of the [NiFe] Dmb variant.

59 BASIC BIOLOGICAL SCIENCES↗

Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins

Radiation of the plant pyridoxal 5'-phosphate (PLP)-dependent aromatic L -amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic L -amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. In this paper, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations.

59 BASIC BIOLOGICAL SCIENCES↗

Glycosyltransferase family 47 (GT47) proteins in plants and animals

Glycosyltransferases (GTs) are carbohydrate-active enzymes that are encoded by the genomes of organisms spanning all domains of life. GTs catalyze glycosidic bond formation, transferring a sugar monomer from an activated donor to an acceptor substrate, often another saccharide. GTs from family 47 (GT47, PF03016) are involved in the synthesis of complex glycoproteins in mammals and insects and play a major role in the synthesis of almost every class of polysaccharide in plants, with the exception of cellulose, callose, and mixed linkage β-1,3/1,4-glucan. GT47 enzymes adopt a GT-B fold and catalyze the formation of glycosidic bonds through an inverting mechanism. Unlike animal genomes, which encode few GT47 enzymes, plant genomes contain 30 or more diverse GT47 coding sequences. With our current knowledge of the GT47 family across plant species brings us an interesting view, showcasing how members exhibit a great diversity in both donor and acceptor substrate specificity, even for members that are classified in the same phylogenetic clade. Thus, we discuss how plant GT47 family members represent a great case to study the relationship between substrate specificity, protein structure, and protein evolution. Most of the plant GT47 enzymes that are identified to date are involved in biosynthesis of plant cell wall polysaccharides, including xyloglucan, xylan, mannan, and pectins. This indicates unique and crucial roles of plant GT47 enzymes in cell wall formation. The aim of this review is to summarize findings about GT47 enzymes and highlight new challenges and approaches on the horizon to study this family.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Enzyme Engineering for Expanded Product Scope (CRADA Final Report)

As part of the Cyclotron Road program, Aralez Inc. investigated methods for producing chemicals sustainably, with a particular focus on engineering enzymes to make novel chemical products or accept new substrates that aren’t found in nature. We used directed evolution and other protein engineering techniques to create enzymes that perform abiological chemistry or make chemical products through a non-natural synthetic route. This work was an important step towards increasing the scope of compounds that can be manufactured in a sustainable fashion, which in turn will alleviate the high energy consumption, costly emissions, and waste that are hallmarks of current methods in chemical manufacturing.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Identification and characterization of substrate- and product-selective nylon hydrolases

Enzymes can rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but few have been shown to hydrolyze synthetic polyamides. Here, in this work, we synthesized and characterized a panel of 95 enzymes from the N-terminal nucleophile hydrolase superfamily with 30%–50% pairwise amino acid identity. We found that nearly 40% of the enzymes had substantial nylon hydrolase activity, but there was no relationship between phylogeny and activity, nor any evidence of prior evolutionary selection for nylon hydrolysis. Several newly identified hydrolases showed substrate selectivity, generating up to 20-fold higher product titers with nylon-6,6 versus nylon-6. However, the yield was still less than 1%, necessitating further optimization before potential applications. Finally, we determined the crystal structure and oligomerization state of a nylon-6,6-selective hydrolase to elucidate structural factors that could affect activity and selectivity. These new enzymes provide insights into nylon hydrolase evolution and opportunities for analysis and engineering of improved hydrolases.

nylon↗