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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Microbes display broad diversity in cobamide preferences

ABSTRACT Cobamides, the vitamin B 12 (cobalamin) family of cofactors, are used by most organisms but produced by only a fraction of prokaryotes, and are thus considered key shared nutrients among microbes. Cobamides are structurally diverse, with multiple different cobamides found in most microbial communities. The ability to use different cobamides has been tested for several bacteria and microalgae, and nearly all show preferences for certain cobamides. This approach is limited by the commercial unavailability of cobamides other than cobalamin. Here, we have extracted and purified seven commercially unavailable cobamides to characterize bacterial cobamide preferences based on growth in specific cobamide-dependent conditions. The tested bacteria include engineered strains of Escherichia coli , Sinorhizobium meliloti , and Bacillus subtilis expressing native or heterologous cobamide-dependent enzymes, cultured under conditions that functionally isolate specific cobamide-dependent processes such as methionine synthesis. Comparison of these results to those of previous studies of diverse bacteria and microalgae revealed that a broad diversity of cobamide preferences exists not only across different organisms but also between different cobamide-dependent metabolic pathways within the same organism. The microbes differed in the cobamides that support growth most efficiently, cobamides that do not support growth, and the minimum cobamide concentrations required for growth. The latter differ by up to four orders of magnitude across organisms from different environments and by up to 20-fold between cobamide-dependent enzymes within the same organism. Given that cobamides are shared, required for use of specific growth substrates, and essential for central metabolism in certain organisms, cobamide preferences likely impact community structure and function. IMPORTANCE Nearly all bacteria are found in microbial communities with tens to thousands of other species. Molecular interactions such as metabolic cooperation and competition are key factors underlying community assembly and structure. Cobamides, the vitamin B 12 family of enzyme cofactors, are one such class of nutrients, produced by only a minority of prokaryotes but required by most microbes. A unique aspect of cobamides is their broad diversity, with nearly 20 structural forms identified in nature. Importantly, this structural diversity impacts growth as most bacteria that have been tested show preferences for specific cobamide forms. We measured cobamide-dependent growth in several model bacteria and compared the results to those of previous analyses of cobamide preference. We found that cobamide preferences vary widely across bacteria, showing the importance of characterizing these aspects of cobamide biology to understand the impact of cobamides on microbial communities.

Mok, Kenny C. (ORCID:0000000252276987)↗
DOE OSTI2024

Pyrophosphate-free glycolysis in Clostridium thermocellum increases both thermodynamic driving force and ethanol titers

Background: Clostridium thermocellum is a promising candidate for production of cellulosic biofuels, however, its final product titer is too low for commercial application, and this may be due to thermodynamic limitations in glycolysis. Previous studies in this organism have revealed a metabolic bottleneck at the phosphofructokinase (PFK) reaction in glycolysis. In the wild-type organism, this reaction uses pyrophosphate (PP i ) as an energy cofactor, which is thermodynamically less favorable compared to reactions that use ATP as a cofactor. Previously we showed that replacing the PP i -linked PFK reaction with an ATP-linked reaction increased the thermodynamic driving force of glycolysis, but only had a local effect on intracellular metabolite concentrations, and did not affect final ethanol titer. Results: In this study, we substituted PP i -pfk with ATP-pfk, deleted the other PPi-requiring glycolytic gene pyruvate:phosphate dikinase (ppdk), and expressed a soluble pyrophosphatase (PPase) and pyruvate kinase (pyk) genes to engineer PP i -free glycolysis in C. thermocellum. We demonstrated a decrease in the reversibility of the PFK reaction, higher levels of lower glycolysis metabolites, and an increase in ethanol titer by an average of 38% (from 15.1 to 21.0 g/L) by using PP i -free glycolysis. Conclusions: By engineering PP i -free glycolysis in C. thermocellum, we achieved an increase in ethanol production. These results demonstrate that optimizing the thermodynamic landscape through metabolic engineering can enhance product titers. While further increases in ethanol titers are necessary for commercial application, this work represents a significant step toward engineering glycolysis in C. thermocellum to increase ethanol titers.

09 BIOMASS FUELS↗
DOE OSTI2020

Data from Stereoconvergent Reduction of Activated Alkenes by a Nicotinamide Free Synergistic Photobiocatalytic System

There is a growing interest in developing cooperative chemoenzymatic reactions to harness the reactivity of chemical catalysts and the selectivity of enzymes for the synthesis of nonracemic chiral compounds. However, existing chemoenzymatic systems with more than one chemical reaction and one enzymatic reaction working cooperatively are rare. Moreover, the application of oxidoreductases in cooperative chemoenzymatic reactions is limited by the necessity of using expensive and unstable redox equivalents such as nicotinamide cofactors. Here, we report a light-driven cooperative chemoenzymatic system comprised of a photoinduced electron transfer reaction (PET) and a photosensitized energy transfer reaction (PEnT) with an enzymatic reduction in one-pot to synthesize chiral building blocks of bioactive compounds. As a proof of concept, ene-reductase was directly regenerated by PET in the absence of external cofactors. Meanwhile, enzymatic reduction worked cooperatively with photocatalyst-catalyzed energy transfer that continuously replenished the reactive isomer from the less reactive one. The whole system stereoconvergently reduced E/Z mixtures of alkenes to the enantiopure products. Additionally, enantioselective enzymatic reduction worked competitively with photocatalyst-catalyzed racemic background reaction and side reactions to channel the overall electron flow to the single enantiopure product. Such a light-driven cooperative chemoenzymatic system holds great potential for asymmetric synthesis using inexpensive petroleum or biomass-derived alkenes.

Catalysis↗
DOE OSTI2023

The Escherichia coli MFS-type transporter genes yhjE, ydiM, and yfcJ are required to produce an active bo3 quinol oxidase

Heme-copper oxygen reductases are membrane-bound oligomeric complexes that are integral to prokaryotic and eukaryotic aerobic respiratory chains. Biogenesis of these enzymes is complex and requires coordinated assembly of the subunits and their cofactors. Some of the components are involved in the acquisition and integration of different heme and copper (Cu) cofactors into these terminal oxygen reductases. As such, MFS-type transporters of the CalT family ( e . g ., CcoA) are required for Cu import and heme-Cu B center biogenesis of the cbb 3 -type cytochrome c oxidases ( cbb 3 -Cox). However, functionally homologous Cu transporters for similar heme-Cu containing bo 3 -type quinol oxidases ( bo 3 -Qox) are unknown. Despite the occurrence of multiple MFS-type transporters, orthologs of CcoA are absent in bacteria like Escherichia coli that contain bo 3 -Qox. In this work, we identified a subset of uncharacterized MFS transporters, based on the presence of putative metal-binding residues, as likely candidates for the missing Cu transporter. Using a genetic approach, we tested whether these transporters are involved in the biogenesis of E . coli bo 3 -Qox. When respiratory growth is dependent on bo 3 -Qox, because of deletion of the bd -type Qox enzymes, three candidate genes, yhjE , ydiM , and yfcJ , were found to be critical for E . coli growth. Radioactive metal uptake assays showed that Δ ydiM has a slower 64 Cu uptake, whereas Δ yhjE accumulates reduced 55 Fe in the cell, while no similar uptake defect is associated with Δ ycfJ . Phylogenomic analyses suggest plausible roles for the YhjE, YdiM, and YfcJ transporters, and overall findings illustrate the diverse roles that the MFS-type transporters play in cellular metal homeostasis and production of active heme-Cu oxygen reductases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Photodamage and Repair in Higher Plant Photosynthesis (Final Technical Report)

Elucidation of the functional properties and structural organization of membrane protein complexes is one of the central objectives of current biochemical investigation. One of the most intriguing aspects of membrane protein function is its role in the mediation of energy transduction in photosynthetic organisms. Light energy, which is the product of a most violent physical process, fusion, is transformed into biological energy equivalents utilized by the photosynthetic cell. This photosynthetic process provides both the carbohydrate that lies at the base of virtually all food chains and, as a byproduct, all of the atmospheric oxygen utilized by heterotrophic organisms. The photosynthetic apparatus, however, is quite susceptible to damage by Reactive Oxygen Species (ROS). These species include O2 ·– , ·OH, H 2 O 2 and 1 O 2 . The production of ROS is an unavoidable byproduct of electron transport under aerobic conditions and these can damage lipids, nucleic acids and proteins. Our research has centered on the identification of amino acid oxidative damage which occurs in the vicinity of photosynthetic energy transfer cofactors. These cofactors participate in excitation energy transfer and linear electron transport within Photosystem II, the Cytochrome b 6 ƒ complex and Photosystem I. Additionally, we have participated in a number of productive collaborations leveraging our expertise in photosynthetic measurements and mass spectrometry to assist other investigators in their research.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Maturation of Rhodobacter capsulatus Multicopper Oxidase CutO Depends on the CopA Copper Efflux Pathway and Requires the cutF Product

Copper (Cu) is an essential cofactor required for redox enzymes in all domains of life. Because of its toxicity, tightly controlled mechanisms ensure Cu delivery for cuproenzyme biogenesis and simultaneously protect cells against toxic Cu. Many Gram-negative bacteria contain extracytoplasmic multicopper oxidases (MCOs), which are involved in periplasmic Cu detoxification. MCOs are unique cuproenzymes because their catalytic center contains multiple Cu atoms, which are required for the oxidation of Cu 1+ to the less toxic Cu 2+ . Hence, Cu is both substrate and essential cofactor of MCOs. Here, we investigated the maturation of Rhodobacter capsulatus MCO CutO and its role in periplasmic Cu detoxification. A survey of CutO activity of R. capsulatus mutants with known defects in Cu homeostasis and in the maturation of the cuproprotein cbb 3 -type cytochrome oxidase ( cbb 3 -Cox) was performed. This revealed that CutO activity is largely independent of the Cu-delivery pathway for cbb 3 -Cox biogenesis, except for the cupric reductase CcoG, which is required for full CutO activity. The most pronounced decrease of CutO activity was observed with strains lacking the cytoplasmic Cu chaperone CopZ, or the Cu-exporting ATPase CopA, indicating that CutO maturation is linked to the CopZ-CopA mediated Cu-detoxification pathway. Our data demonstrate that CutO is important for cellular Cu resistance under both aerobic and anaerobic growth conditions. CutO is encoded in the cutFOG operon, but only CutF, and not CutG, is essential for CutO activity. No CutO activity is detectable when cutF or its putative Cu-binding motif are mutated, suggesting that the cutF product serves as a Cu-binding component required for active CutO production. Bioinformatic analyses of CutF-like proteins support their widespread roles as putative Cu-binding proteins for several Cu-relay pathways. Our overall findings show that the cytoplasmic CopZ-CopA dependent Cu detoxification pathway contributes to providing Cu to CutO maturation, a process that strictly relies on cutF .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

An Abundant and Diverse New Family of Electron Bifurcating Enzymes With a Non-canonical Catalytic Mechanism

Microorganisms utilize electron bifurcating enzymes in metabolic pathways to carry out thermodynamically unfavorable reactions. Bifurcating FeFe-hydrogenases (HydABC) reversibly oxidize NADH (E′∼−280 mV, under physiological conditions) and reduce protons to H 2 gas (E°′−414 mV) by coupling this endergonic reaction to the exergonic reduction of protons by reduced ferredoxin (Fd) (E′∼−500 mV). We show here that HydABC homologs are surprisingly ubiquitous in the microbial world and are represented by 57 phylogenetically distinct clades but only about half are FeFe-hydrogenases. The others have replaced the hydrogenase domain with another oxidoreductase domain or they contain additional subunits, both of which enable various third reactions to be reversibly coupled to NAD + and Fd reduction. We hypothesize that all of these enzymes carry out electron bifurcation and that their third substrates can include hydrogen peroxide, pyruvate, carbon monoxide, aldehydes, aryl-CoA thioesters, NADP + , cofactor F 420 , formate, and quinones, as well as many yet to be discovered. Some of the enzymes are proposed to be integral membrane-bound proton-translocating complexes. These different functionalities are associated with phylogenetically distinct clades and in many cases with specific microbial phyla. We propose that this new and abundant class of electron bifurcating enzyme be referred to as the Bfu family whose defining feature is a conserved bifurcating BfuBC core. This core contains FMN and six iron sulfur clusters and it interacts directly with ferredoxin (Fd) and NAD(H). Electrons to or from the third substrate are fed into the BfuBC core via BfuA. The other three known families of electron bifurcating enzyme (abbreviated as Nfn, EtfAB, and HdrA) contain a special FAD that bifurcates electrons to high and low potential pathways. The Bfu family are proposed to use a different electron bifurcation mechanism that involves a combination of FMN and three adjacent iron sulfur clusters, including a novel [2Fe-2S] cluster with pentacoordinate and partial non-Cys coordination. The absolute conservation of the redox cofactors of BfuBC in all members of the Bfu enzyme family indicate they have the same non-canonical mechanism to bifurcate electrons. A hypothetical catalytic mechanism is proposed as a basis for future spectroscopic analyses of Bfu family members.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Rational design of photosynthetic reaction center protein maquettes

New technologies for efficient solar-to-fuel energy conversion will help facilitate a global shift from dependence on fossil fuels to renewable energy. Nature uses photosynthetic reaction centers to convert photon energy into a cascade of electron-transfer reactions that eventually produce chemical fuel. The design of new reaction centers de novo deepens our understanding of photosynthetic charge separation and may one day allow production of biofuels with higher thermodynamic efficiency than natural photosystems. Recently, we described the multi-step electron-transfer activity of a designed reaction center maquette protein (the RC maquette), which can assemble metal ions, tyrosine, a Zn tetrapyrrole, and heme into an electron-transport chain. Here, we detail our modular strategy for rational protein design and show that the intended RC maquette design agrees with crystal structures in various states of assembly. A flexible, dynamic apo-state collapses by design into a more ordered holo-state upon cofactor binding. Crystal structures illustrate the structural transitions upon binding of different cofactors. Spectroscopic assays demonstrate that the RC maquette binds various electron donors, pigments, and electron acceptors with high affinity. We close with a critique of the present RC maquette design and use electron-tunneling theory to envision a path toward a designed RC with a substantially higher thermodynamic efficiency than natural photosystems.

09 BIOMASS FUELS↗
DOE OSTI2023

Arsenic Exposure Causes Global Changes in the Metalloproteome of Escherichia coli

Arsenic is a toxic metalloid with differential biological effects, depending on speciation and concentration. Trivalent arsenic (arsenite, AsIII) is more toxic at lower concentrations than the pentavalent form (arsenate, AsV). In E. coli, the proteins encoded by the arsRBC operon are the major arsenic detoxification mechanism. Our previous transcriptional analyses indicate broad changes in metal uptake and regulation upon arsenic exposure. Currently, it is not known how arsenic exposure impacts the cellular distribution of other metals. This study examines the metalloproteome of E. coli strains with and without the arsRBC operon in response to sublethal doses of AsIII and AsV. Size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICPMS) was used to investigate the distribution of five metals (56Fe, 24Mg, 66Zn, 75As, and 63Cu) in proteins and protein complexes under native conditions. Parallel analysis by SEC-UV-Vis spectroscopy monitored the presence of protein cofactors. Together, these data reveal global changes in the metalloproteome, proteome, protein cofactors, and soluble intracellular metal pools in response to arsenic stress in E. coli. This work brings to light one outcome of metal exposure and suggests that metal toxicity on the cellular level arises from direct and indirect effects.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Harnessing photoenzymatic reactions for unnatural biosynthesis in microorganisms

Photoenzymatic catalysis enables new-to-nature transformations, but its scalability is limited by high enzyme loading, costly cofactors, and radical-induced instability. Here we report the integration of light-driven photoenzymatic reactions into the cellular metabolism of Escherichia coli, bridging flavin-based photobiocatalysis with biosynthesis. Using synthetic biology strategies, we engineered microbial cells to continuously produce olefin substrates and ene-reductase photoenzyme while regenerating cofactors directly from glucose. By externally supplying radical precursors or by introducing synthetic pathways for their in situ production, we enabled fermentation-based microbial photobiosynthesis, achieving high titers and demonstrating its feasibility for scale-up in bioreactor. This approach extends photobiocatalysis from in vitro applications to in vivo semi-biosynthesis and complete biosynthesis, revealing its full potential for integrating light-driven reactions into cellular metabolism.

Bioproducts↗
DOE OSTI2024

Employing bacterial microcompartments to create privileged redox pools for biofuel production (Final report: Accomplishments)

Over the award period we made progress on all three objectives. We completed our goal of understanding the impact of cofactor recycling on pathway performance for a propanediol-related pathway (Objective 1) and coupled this data with kinetic metabolic models developed in Objective 3, resulting in several products (4 published research articles, 1 review and 3 other research manuscripts in preparation). We completed half of Objective 2 but found the pathway was incompatible with compartmentalization, which is an important but unexpected result. The details are relayed in the following sections, grouped by objective and task as applicable.

09 BIOMASS FUELS↗
DOE OSTI2026

The HypA and HypB metallochaperones from Methanococcus maripaludis have unique metal-binding properties and a distinct nickel transfer mechanism

[NiFe] hydrogenases are widespread microbial metalloenzymes that catalyze the reversible conversion of hydrogen (H2) to protons and electrons, playing key roles in energy metabolism. The biosynthesis of the NiFe(CN) 2 CO cofactor involves a suite of maturation proteins, including the HypA and HypB nickel metallochaperones. Here, we define the metal-binding properties, nucleotide-dependent behavior, and functional interplay of HypA and HypB from the hydrogenotrophic methanogenic archaeon, Methanococcus maripaludis . Methanogens have multiple essential nickel-dependent enzymes, so they require efficient systems for nickel delivery that remain largely unexplored. Purified M. maripaludis HypA binds zinc or mononuclear iron at the C-terminal metal binding site, the latter of which has not been reported in other HypA proteins and may serve a unique regulatory role in methanogens. The G-protein metallochaperone HypB binds nickel at the G-domain, which stimulates GTPase activity. Size exclusion chromatography experiments reveal that HypA and HypB form complexes in the presence of nickel, and zinc-bound HypA is optimized for nickel transfer from HypB. The identity of the nucleotide bound to HypB (GDP or GTP) alters the oligomeric state of HypA-HypB complexes, supporting a GTPase-mediated nickel delivery pathway. The HypA-HypB 2 complex configuration is enriched and stable in the presence of GDP and nickel, indicating that this complex delivers nickel to the hydrogenase as opposed to HypA alone. Interestingly, affinity purification-mass spectrometry revealed that HypB interacts with several nickel-dependent proteins, suggesting that HypB may play a broader role in nickel homeostasis in M. maripaludis . Together, this work establishes a biochemical framework for HypAB-mediated nickel trafficking in methanogens.

[NiFe] hydrogenase↗
DOE OSTI2024

Soil microbial community response to corrinoids is shaped by a natural reservoir of vitamin B12

Abstract Soil microbial communities perform critical ecosystem services through the collective metabolic activities of numerous individual organisms. Most microbes use corrinoids, a structurally diverse family of cofactors related to vitamin B12. Corrinoid structure influences the growth of individual microbes, yet how these growth responses scale to the community level remains unknown. Analysis of metagenome-assembled genomes suggests that corrinoids are supplied to the community by members of the archaeal and bacterial phyla Thermoproteota, Actinobacteria, and Proteobacteria. Corrinoids were found largely adhered to the soil matrix in a grassland soil, at levels exceeding those required by cultured bacteria. Enrichment cultures and soil microcosms seeded with different corrinoids showed distinct shifts in bacterial community composition, supporting the hypothesis that corrinoid structure can shape communities. Environmental context influenced both community- and taxon-specific responses to specific corrinoids. These results implicate corrinoids as key determinants of soil microbiome structure and suggest that environmental micronutrient reservoirs promote community stability.

58 GEOSCIENCES↗
DOE OSTI2023

New structures of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis provide a framework for a novel catalytic mechanism for the entire class

Class II Fructose-1,6-bisphosphatases (FBPaseII) (EC: 3.1.3.11) are highly conserved essential enzymes in the gluconeogenic pathway of microorganisms. Previous crystallographic studies of FBPasesII provided insights into various inactivated states of the enzyme in different species. Presented here is the first crystal structure of FBPaseII in an active state, solved for the enzyme from Francisella tularensis (FtFBPaseII), containing native metal cofactor Mn 2+ and complexed with catalytic product fructose-6-phosphate (F6P). Another crystal structure of the same enzyme complex is presented in the inactivated state due to the structural changes introduced by crystal packing. Analysis of the interatomic distances among the substrate, product, and divalent metal cations in the catalytic centers of the enzyme led to a revision of the catalytic mechanism suggested previously for class II FBPases. We propose that phosphate-1 is cleaved from the substrate fructose-1,6-bisphosphate (F1,6BP) by T89 in a proximal α-helix backbone (G88-T89-T90-I91-T92-S93-K94) in which the substrate transition state is stabilized by the positive dipole of the $\langle$-helix backbone. Once cleaved a water molecule found in the active site liberates the inorganic phosphate from T89 completing the catalytic mechanism. Additionally, a crystal structure of Mycobacterium tuberculosis FBPaseII (MtFBPaseII) containing a bound F1,6BP is presented to further support the substrate binding and novel catalytic mechanism suggested for this class of enzymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Combinatorial library design for improving isobutanol production in Saccharomyces cerevisiae

Saccharomyces cerevisiae is the dominant fermentative producer of ethanol in industry and a preferred host for production of other biofuels. That said, rewiring the metabolism of S. cerevisiae to produce other fermentation products, such as isobutanol, remains an academic challenge. Many studies report aerobic production of isobutanol, but ethanol remains a substantial by-product under these conditions due to the Crabtree effect. These studies indicate that the native isobutanol pathway is incapable of carrying sufficient flux to displace ethanol. In this report, we screened a combinatorial library of pathway enzymes to identify an isobutanol pathway cassette capable of supporting the growth of a non-ethanol producing S. cerevisiae . We began by identifying a diverse set of isobutanol pathway enzyme homologs and combined each open reading frame with varied-strength promoters in a combinatorial, pooled fashion. We applied a growth-coupled screen where a functional isobutanol pathway restored NAD + regeneration during glucose catabolism that is otherwise repressed via the Crabtree effect. Using this screen, we isolated a cassette consisting of a mosaic of bacterial and cytosol-localized fungal enzymes that conferred under aerobic conditions the ability to produce 364 mg/L isobutanol (8.8% of the theoretical maximum yield). We next shifted the cofactor usage of the isolated ketol-acid reductoisomerase enzyme in the cassette from NADPH to NADH-preferring to improve redox balance. The approach used herein isolated isobutanol producing strains that approach the best in the literature without producing substantial ethanol titers. Still, the best isolated cassette was insufficient to support anaerobic growth in the absence of ethanol fermentation - indicating the presence of further fundamental gaps in our understanding of yeast fermentation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3′ terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The crystal structure of methanogen McrD , a methyl‐coenzyme M reductase‐associated protein

Methyl‐coenzyme M reductase (MCR) is a multi‐subunit (α 2 β 2 γ 2 ) enzyme responsible for methane formation via its unique F 430 cofactor. The genes responsible for producing MCR ( mcrA , mcrB and mcrG ) are typically colocated with two other highly conserved genes mcrC and mcrD . We present here the high‐resolution crystal structure for McrD from a human gut methanogen Methanomassiliicoccus luminyensis strain B10. The structure reveals that McrD comprises a ferredoxin‐like domain assembled into an α + β barrel‐like dimer with conformational flexibility exhibited by a functional loop. The description of the M. luminyensis McrD crystal structure contributes to our understanding of this key conserved methanogen protein typically responsible for promoting MCR activity and the production of methane, a greenhouse gas.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

[FeFe]‐Hydrogenase In Vitro Maturation

Abstract The [FeFe]‐hydrogenase H‐cluster is a complex organometallic cofactor whose assembly and installation requires three dedicated accessory proteins referred to as HydE, HydF, and HydG. The roles of these maturases and the precise mechanisms by which they synthesize and insert the H‐cluster are not fully understood. This Minireview will focus on new insights into the [FeFe]‐hydrogenase maturation process that have been provided by in vitro approaches in which the biosynthetic pathway has been partially or fully reconstructed using semisynthetic and enzyme‐based approaches. Specifically, the application of these in vitro, semisynthetic, and fully defined approaches has shed light on the roles of individual maturation enzymes, the nature of H‐cluster assembly intermediates, the molecular precursors of H‐cluster ligands, and the sequence of steps involved in [FeFe]‐hydrogenase maturation.

Pagnier, Adrien↗