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DOE OSTI2022

[Fe 4 S 4 ] cubane in sulfite reductases: new insights into bonding properties and reactivity

The dissimilatory sulfite reductase enzyme has very characteristic active site where the substrate binds to an iron site, ligated by a siroheme macrocycle and a thiol directly connected to a [Fe 4 S 4 ] cluster. This arrangement gives the enzyme remarkable efficiency in reducing sulfite and nitrite all the way to hydrogen sulfide and ammonia. For the first time we present a theoretical study where substrate binding modalities and activation are elucidated using active site models containing proton supply side chains and the [Fe 4 S 4 ] cluster. Density functional theory (DFT) was deployed in conjunction with the energy decomposition scheme (as implemented in AMS), the quantum theory of atoms in molecules (QTAIM), and conceptual DFT (cDFT) descriptors. We quantified the role of the electrostatic interactions inside the active site created by the side chains as well as the influence of the [Fe4S4] cluster on the substrate binding. Furthermore, using conceptual DFT results we shed light of the activation process, thus, laying foundation for further mechanistic studies. We found that the bonding of the ligands to the iron complex is dominated by electrostatic interactions, but the presence of the [Fe 4 S 4 ] cubane leads to substantial changes in electronic interaction. The spin state of the cubane, however, affects the binding energy only marginally. The conceptual DFT results show that the presence of the [Fe 4 S 4 ] cubane affects the reactivity of the active site as it is involved in electron transfer. This is corroborated by an increase in the electrophilicity index, thus making the active site more prone to react with the ligands. The interaction energies between the ligand and the siroheme group are also increased upon the presence of the cubane group, thus, suggesting that the siroheme group is not an innocent spectator but plays an active role in the reactivity of the dSIR active site.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Cell-free synthetic biology for natural product biosynthesis and discovery

Natural products have applications as biopharmaceuticals, agrochemicals, and other high-value chemicals. However, there are challenges in isolating natural products from their native producers (e.g. bacteria, fungi, plants). In many cases, synthetic chemistry or heterologous expression must be used to access these important molecules. The biosynthetic machinery to generate these compounds is found within biosynthetic gene clusters, primarily consisting of the enzymes that biosynthesise a range of natural product classes (including, but not limited to ribosomal and nonribosomal peptides, polyketides, and terpenoids). Cell-free synthetic biology has emerged in recent years as a bottom-up technology applied towards both prototyping pathways and producing molecules. Recently, it has been applied to natural products, both to characterise biosynthetic pathways and produce new metabolites. This review discusses the core biochemistry of cell-free synthetic biology applied to metabolite production and critiques its advantages and disadvantages compared to whole cell and/or chemical production routes. Specifically, we review the advances in cell-free biosynthesis of ribosomal peptides, analyse the rapid prototyping of natural product biosynthetic enzymes and pathways, highlight advances in novel antimicrobial discovery, and discuss the rising use of cell-free technologies in industrial biotechnology and synthetic biology.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Simultaneous occupancy of Cu C and Cu D in the ammonia monooxygenase active site

Ammonia monooxygenase (AMO), a copper-dependent membrane enzyme, catalyzes the first and rate-limiting step of nitrification: the oxidation of ammonia to hydroxylamine. Despite its central role in the global nitrogen cycle and its biotechnological relevance, structural characterization of AMO has lagged behind that of its homolog, particulate methane monooxygenase (pMMO), due to the slow growth rates of ammonia-oxidizing bacteria and the instability of AMO upon purification. Recent cryoEM studies of Nitrosomonas europaea AMO and Methylococcus capsulatus (Bath) pMMO in native membranes revealed new structural features, including two adjacent copper-binding sites in the transmembrane region, Cu C and Cu D , believed to constitute the active site. Although multiple structures were determined under various conditions, simultaneous occupancy of Cu C and Cu D was never observed, leaving their potential functional interplay unresolved. Here we report the 2.6 Å resolution cryoEM structure of AMO from Nitrosospira briensis C-128 in isolated native membranes. This structure reveals the first instance of simultaneous copper occupancy of the Cu C and Cu D sites, along with occupancy of the periplasmic Cu B site. Electron paramagnetic resonance (EPR) spectroscopic data indicate that the Cu B site is primarily occupied by Cu(II), while Cu C and Cu D are primarily occupied by diamagnetic ions, presumably Cu(I). Notably, a lipid molecule is bound between the Cu C and Cu D sites, separating them by ∼8.0 Å. The results underscore the importance of studying these enzymes in their native environments across species to resolve conserved and divergent molecular features.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Mutation of active site glutamate in serine hydroxymethyltransferase allows trapping a reactive intermediate: a combined neutron and X-ray crystallography study

Serine hydroxymethyltransferase (SHMT) is a pyridoxal-5′-phosphate (PLP) dependent enzyme that catalyzes a chemical transformation essential for the one-carbon (1C) metabolism. SHMT reversibly converts L-Ser into Gly and transfers a 1C unit to tetrahydrofolate (THF) to give 5,10-methylene-THF (5,10-MTHF). 5,10-MTHF, a 1C-unit donor, plays a crucial role in the downstream biomolecular syntheses required for the cell homeostasis and proliferation. SHMT is a prominent target for the drug discovery to battle bacterial and parasitic infections, and to treat various types of cancer. SHMT-catalyzed chemistry is governed by the general acid-base catalysis. Knowledge of the catalytic mechanism can aid drug design but can only be achieved when the atomic details of each reaction step are mapped, including accurate determination of hydrogen atom positions. Here we utilized the inactive E53Q mutant of Thermus thermophilus ( Tth ) SHMT to directly determine protonation states with room-temperature neutron crystallography and to capture a reactive intermediate containing the PLP-L-Ser external aldimine and THF in the enzyme active site. We observed protonation of the Schiff base nitrogen (N SB ) in the PLP internal aldimine but no change in the protonation states of other ionizable PLP groups and active site residues compared to wild-type Tth SHMT. X-ray structural analysis of the ternary intermediate complex E53Q-Ser-THF that eluded previous structural characterization shows the strategic positioning of the E53Q side chain in close proximity to the external aldimine and THF and reinforces the proposed role for E53 as the driver of proton transfer events along the reaction pathway.

Drago, Victoria N. [Oak Ridge National Laboratory ↗
DOE OSTI2020

Insights into the unique carboxylation reactions in the metabolism of propylene and acetone

Alkenes and ketones are two classes of ubiquitous, toxic organic compounds in natural environments produced in several biological and anthropogenic processes. In spite of their toxicity, these compounds are utilized as primary carbon and energy sources or are generated as intermediate metabolites in the metabolism of other compounds by many diverse bacteria. The aerobic metabolism of some of the smallest and most volatile of these compounds (propylene, acetone, isopropanol) involves novel carboxylation reactions resulting in a common product acetoacetate. Propylene is metabolized in a four-step pathway involving five enzymes where the penultimate step is a carboxylation reaction catalyzed by a unique disulfide oxidoreductase that couples reductive cleavage of a thioether linkage with carboxylation to produce acetoacetate. The carboxylation of isopropanol begins with conversion to acetone via an alcohol dehydrogenase. Acetone is converted to acetoacetate in a single step by an acetone carboxylase which couples the hydrolysis of MgATP to the activation of both acetone and bicarbonate, generating highly reactive intermediates that are condensed into acetoacetate at a Mn2+ containing the active site. Acetoacetate is then utilized in central metabolism where it is readily converted to acetyl-coenzyme A and subsequently converted into biomass or utilized in energy metabolism via the tricarboxylic acid cycle. This review summarizes recent structural and biochemical findings that have contributed significant insights into the mechanism of these two unique carboxylating enzymes.

Biochemistry & Molecular Biology↗
DOE OSTI2020

Bioinformatic and experimental evidence for suicidal and catalytic plant THI4s

Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsistz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC–MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mechanism in planta. Unexpectedly, transcriptome data mining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in developing grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thiazole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain-filling period.

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

Overexpression of phospholipid: diacylglycerol acyltransferase in Brassica napus results in changes in lipid metabolism and oil accumulation

The regulation of lipid metabolism in oil seeds is still not fully understood and increasing our knowledge in this regard is of great economic, as well as intellectual, importance. Oilseed rape (Brassica napus) is a major global oil crop where increases in triacylglycerol (TAG) accumulation have been achieved by overexpression of relevant biosynthetic enzymes. In this study, we expressed Arabidopsis phospholipid: diacylglycerol acyltransferase (PDAT1), one of the two major TAG-forming plant enzymes in B. napus DH12075 to evaluate its effect on lipid metabolism in developing seeds and to estimate its flux control coefficient. Despite several-fold increase in PDAT activity, seeds of three independently generated PDAT transgenic events showed a small but consistent decrease in seed oil content and had altered fatty acid composition of phosphoglycerides and TAG, towards less unsaturation. Mass spectrometry imaging of seed sections confirmed the shift in lipid compositions and indicated that PDAT overexpression altered the distinct heterogeneous distributions of phosphatidylcholine (PC) molecular species. Similar, but less pronounced, changes in TAG molecular species distributions were observed. Our data indicate that PDAT exerts a small, negative, flux control on TAG biosynthesis and could have under-appreciated effects in fine-tuning of B. napus seed lipid composition in a tissue-specific manner. This has important implications for efforts to increase oil accumulation in similar crops.

Biochemistry & Molecular Biology↗
DOE OSTI2025

Structural and kinetic characterization of an acetoacetyl-Coenzyme A: acetate Coenzyme A transferase from the extreme thermophile Thermosipho melanesiensis

Family 1 Coenzyme A transferases (CtfAB) from the extremely thermophilic bacterium, Thermosipho melanesiensis, has been used for in vivo acetone production up to 70°C. This enzyme has tentatively been identified as the rate-limiting step, due to its relatively low-binding affinity for acetate. However, existing kinetic and mechanistic studies on this enzyme are insufficient to evaluate this hypothesis. Here, kinetic analysis of purified recombinant T. melanesiensis CtfAB showed that it has a ping-pong bi-bi mechanism typical of Coenzyme A (CoA) transferases with Km values for acetate and acetoacetyl-CoA of 85 mM and 135 μM, respectively. Product inhibition by acetyl-CoA was competitive with respect to acetoacetyl-CoA and non-competitive with respect to acetate. Crystal structures of wild-type and mutant T. melanesiensis CtfAB were solved in the presence of acetate and in the presence or absence of acetyl-CoA. These structures led to a proposed structural basis for the competitive and non-competitive inhibition of acetyl-CoA: acetate binds independently of acetyl-CoA in an apparent low-affinity binding pocket in CtfA that is directly adjacent to a catalytic glutamate in CtfB. Similar to other CoA transferases, acetyl-CoA is bound in an apparent high-affinity binding site in CtfB with most interactions occurring between the phospho-ADP of CoA and CtfB residues far from the acetate binding pocket. This structural-based mechanism also explains the organic acid promiscuity of CtfAB. High-affinity interactions are predominantly between the conserved phospho-ADP of CoA, and the variable organic acid binding site is a low-affinity binding site with few specific interactions.

Biochemistry & Molecular Biology↗
DOE OSTI2020

Arabidopsis Dehiscence Zone Polygalacturonase 1 (ADPG1) releases latent defense signals in stems with reduced lignin content

There is considerable interest in engineering plant cell wall components, particularly lignin, to improve forage quality and biomass properties for processing to fuels and bioproducts. However, modifying lignin content and/or composition in transgenic plants through down-regulation of lignin biosynthetic enzymes can induce expression of defense response genes in the absence of biotic or abiotic stress. Arabidopsis thaliana lines with altered lignin through down-regulation of hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) or loss of function of cinnamoyl CoA reductase 1 (CCR1) express a suite of pathogenesis-related (PR) protein genes. The plants also exhibit extensive cell wall remodeling associated with induction of multiple cell wall-degrading enzymes, a process which renders the corresponding biomass a substrate for growth of the cellulolytic thermophile Caldicellulosiruptor bescii lacking a functional pectinase gene cluster. The cell wall remodeling also results in the release of size- and charge-heterogeneous pectic oligosaccharide elicitors of PR gene expression. Genetic analysis shows that both in planta PR gene expression and release of elicitors are the result of ectopic expression in xylem of the gene Arabidopsis Dehiscence Zone Polygalacturonase 1 (ADPG1), which is normally expressed during anther and silique dehiscence. These data highlight the importance of pectin in cell wall integrity and the value of lignin modification as a tool to interrogate the informational content of plant cell walls.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structure of the cytochrome aa 3 -600 heme-copper menaquinol oxidase bound to inhibitor HQNO shows TM0 is part of the quinol binding site

Virtually all proton-pumping terminal respiratory oxygen reductases are members of the heme-copper oxidoreductase superfamily. Most of these enzymes use reduced cytochrome c as a source of electrons, but a group of enzymes have evolved to directly oxidize membrane-bound quinols, usually menaquinol or ubiquinol. All of the quinol oxidases have an additional transmembrane helix (TM0) in subunit I that is not present in the related cytochrome c oxidases. The current work reports the 3.6-Å-resolution X-ray structure of the cytochrome aa 3 -600 menaquinol oxidase from Bacillus subtilis containing 1 equivalent of menaquinone. The structure shows that TM0 forms part of a cleft to accommodate the menaquinol-7 substrate. Crystals which have been soaked with the quinol-analog inhibitor HQNO (N-oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveal a single binding site where the inhibitor forms hydrogen bonds to amino acid residues shown previously by spectroscopic methods to interact with the semiquinone state of menaquinone, a catalytic intermediate.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Targeted inhibition of gut bacterial β-glucuronidase activity enhances anticancer drug efficacy

Irinotecan treats a range of solid tumors, but its effectiveness is severely limited by gastrointestinal (GI) tract toxicity caused by gut bacterial β-glucuronidase (GUS) enzymes. Targeted bacterial GUS inhibitors have been shown to partially alleviate irinotecan-induced GI tract damage and resultant diarrhea in mice. Here, we unravel the mechanistic basis for GI protection by gut microbial GUS inhibitors using in vivo models. We use in vitro, in fimo, and in vivo models to determine whether GUS inhibition alters the anticancer efficacy of irinotecan. We demonstrate that a single dose of irinotecan increases GI bacterial GUS activity in 1 d and reduces intestinal epithelial cell proliferation in 5 d, both blocked by a single dose of a GUS inhibitor. In a tumor xenograft model, GUS inhibition prevents intestinal toxicity and maintains the antitumor efficacy of irinotecan. Remarkably, GUS inhibitor also effectively blocks the striking irinotecan-induced bloom of Enterobacteriaceae in immune-deficient mice. In a genetically engineered mouse model of cancer, GUS inhibition alleviates gut damage, improves survival, and does not alter gut microbial composition; however, by allowing dose intensification, it dramatically improves irinotecan’s effectiveness, reducing tumors to a fraction of that achieved by irinotecan alone, while simultaneously promoting epithelial regeneration. These results indicate that targeted gut microbial enzyme inhibitors can improve cancer chemotherapeutic outcomes by protecting the gut epithelium from microbial dysbiosis and proliferative crypt damage.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Structural basis of peptidoglycan endopeptidase regulation

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae. Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The roles of SDHAF2 and dicarboxylate in covalent flavinylation of SDHA, the human complex II flavoprotein

Mitochondrial complex II, also known as succinate dehydrogenase (SDH), is an integral-membrane heterotetramer (SDHABCD) that links two essential energy-producing processes, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. A significant amount of information is available on the structure and function of mature complex II from a range of organisms. However, there is a gap in our understanding of how the enzyme assembles into a functional complex, and disease-associated complex II insufficiency may result from incorrect function of the mature enzyme or from assembly defects. Here, we investigate the assembly of human complex II by combining a biochemical reconstructionist approach with structural studies. We report an X-ray structure of human SDHA and its dedicated assembly factor SDHAF2. Importantly, we also identify a small molecule dicarboxylate that acts as an essential cofactor in this process and works in synergy with SDHAF2 to properly orient the flavin and capping domains of SDHA. This reorganizes the active site, which is located at the interface of these domains, and adjusts the pK a of SDHA R451 so that covalent attachment of the flavin adenine dinucleotide (FAD) cofactor is supported. We analyze the impact of disease-associated SDHA mutations on assembly and identify four distinct conformational forms of the complex II flavoprotein that we assign to roles in assembly and catalysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Structural elucidation of the cis -prenyltransferase NgBR/DHDDS complex reveals insights in regulation of protein glycosylation

Cis-prenyltransferase (cis-PTase) catalyzes the rate-limiting step in the synthesis of glycosyl carrier lipids required for protein glycosylation in the lumen of endoplasmic reticulum. Here, we report the crystal structure of the human NgBR/DHDDS complex, which represents an atomic resolution structure for any heterodimeric cis-PTase. The crystal structure sheds light on how NgBR stabilizes DHDDS through dimerization, participates in the enzyme’s active site through its C-terminal -RXG- motif, and how phospholipids markedly stimulate cis-PTase activity. Comparison of NgBR/DHDDS with homodimeric cis-PTase structures leads to a model where the elongating isoprene chain extends beyond the enzyme’s active site tunnel, and an insert within the α3 helix helps to stabilize this energetically unfavorable state to enable long-chain synthesis to occur. These data provide unique insights into how heterodimeric cis-PTases have evolved from their ancestral, homodimeric forms to fulfill their function in long-chain polyprenol synthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The antibiotic sorangicin A inhibits promoter DNA unwinding in a Mycobacterium tuberculosis rifampicin-resistant RNA polymerase

Rifampicin (Rif) is a first-line therapeutic used to treat the infectious disease tuberculosis (TB), which is caused by the pathogen Mycobacterium tuberculosis (Mtb). The emergence of Rif-resistant (Rif R ) Mtb presents a need for new antibiotics. Rif targets the enzyme RNA polymerase (RNAP). Sorangicin A (Sor) is an unrelated inhibitor that binds in the Rif-binding pocket of RNAP. Sor inhibits a subset of Rif R RNAPs, including the most prevalent clinical Rif R RNAP substitution found in Mtb infected patients (S456>L of the β subunit). In this paper, we present structural and biochemical data demonstrating that Sor inhibits the wild-type Mtb RNAP by a similar mechanism as Rif: by preventing the translocation of very short RNAs. By contrast, Sor inhibits the RifR S456L enzyme at an earlier step, preventing the transition of a partially unwound promoter DNA intermediate to the fully opened DNA and blocking the template-strand DNA from reaching the active site in the RNAP catalytic center. By defining template-strand blocking as a mechanism for inhibition, we provide a mechanistic drug target in RNAP. Our finding that Sor inhibits the wild-type and mutant RNAPs through different mechanisms prompts future considerations for designing antibiotics against resistant targets. Also, we show that Sor has a better pharmacokinetic profile than Rif, making it a suitable starting molecule to design drugs to be used for the treatment of TB patients with comorbidities who require multiple medications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Cryoelectron microscopy structure and mechanism of the membrane-associated electron-bifurcating flavoprotein Fix/EtfABCX

Significance Electron bifurcation is a recently recognized mechanism of biological energy conservation that is widespread in anaerobic microorganisms and provides low-potential, high-energy electrons to drive otherwise nonspontaneous metabolic reactions. Here we describe the structure of a membrane-associated bifurcating enzyme from an aerobic microorganism. Termed Fix/EtfABCX, this enzyme is used by some nitrogen-fixing microbes to drive the low-potential reduction of nitrogen gas to ammonia. Fix/EtfABCX is a superdimer and a catalytic mechanism is proposed involving six flavins, two of which bifurcate, four iron-sulfur clusters, and two menaquinones. Fix/EtfABCX shares structural similarity with mammalian quinone oxidoreductase involved in mitochondrial fatty acid oxidation, suggesting an unexpected evolutionary link between bifurcating and nonbifurcating systems.

59 BASIC BIOLOGICAL SCIENCES↗