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| Open AccessA three-level regulatory mechanism of the aldo-keto reductase subfamily AKR12D
Here, the authors characterise an aldo-keto reductase AKRtyl, which belongs to a previously unidentified subfamily AKR12D. They uncover a complex mechanism of allosteric regulation that is mediated by 3 distinct states.
- Zhihong Xiao
- , Jinyin Zha
- & Shaobo Dai
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Article
| Open AccessUnderstanding activity-stability tradeoffs in biocatalysts by enzyme proximity sequencing
Understanding the complex relationships between enzyme sequence, folding stability and catalytic activity is essential for applications, but current technologies cannot simultaneously resolve both stability and activity phenotypes and couple these to gene sequences at large scale. Here, the authors report Enzyme Proximity Sequencing (EP-Seq), a deep mutational scanning method to assay both expression level and catalytic activity of thousands of oxidoreductase variants from a cellular pool in a single experiment.
- Rosario Vanella
- , Christoph Küng
- & Michael A. Nash
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Article
| Open AccessDeciphering the mechanism of glutaredoxin-catalyzed roGFP2 redox sensing reveals a ternary complex with glutathione for protein disulfide reduction
Fusion proteins between roGFP2 and glutaredoxins are used for intracellular redox measurements. Here, the authors determined all rate constants of the reaction cycle for roGFP2 measurements and identified an alternative glutaredoxin mechanism.
- Fabian Geissel
- , Lukas Lang
- & Marcel Deponte
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Article
| Open AccessUsing the heme peroxidase APEX2 to probe intracellular H2O2 flux and diffusion
Previous genetically encoded H2O2 probes are based on reversible thiol oxidation. Here, a heme peroxidase is introduced as a thiol-independent H2O2 probe. APEX2 converts H2O2 into fluorescent or luminescent signals, allowing its quantification.
- Mohammad Eid
- , Uladzimir Barayeu
- & Tobias P. Dick
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Article
| Open AccessA genome-scale metabolic model of parasitic whipworm
In this work, Bay et al describe the construction of the first genome-scale metabolic model for the parasitic whipworm, Trichuris muris and use it to identify novel metabolic pathways and predict critical enzymes and essential metabolites for worm survival.
- Ömer F. Bay
- , Kelly S. Hayes
- & Ian S. Roberts
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Article
| Open AccessLactate dehydrogenase D is a general dehydrogenase for D-2-hydroxyacids and is associated with D-lactic acidosis
Currently the structure and biological function of Lactate Dehydrogenase D (LDHD) are unclear. Here the authors report the structure of LDHD bound with various ligands and show that LDHD is a general dehydrogenase for D-2-hydroxyacids with small to moderate-size hydrophobic moieties and investigate loss-of-function mutations that play an important role in D-lactic acidosis.
- Shan Jin
- , Xingchen Chen
- & Jianping Ding
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Article
| Open AccessTriepoxide formation by a flavin-dependent monooxygenase in monensin biosynthesis
MonCI, a flavin-dependent monooxygenase, transforms all three C = C groups in the polyene substrate into epoxides during monensin A biosynthesis. Here, the authors present the structural basis for this enzyme’s regio- and stereoselective epoxidation activity.
- Qian Wang
- , Ning Liu
- & Chu-Young Kim
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Article
| Open AccessStructural and functional fine mapping of cysteines in mammalian glutaredoxin reveal their differential oxidation susceptibility
Glutaredoxin (GLRX) is a key enzyme in redox regulation via oxidation of protein cysteines and its activity is disrupted in diverse human diseases. This study integrates molecular modeling and biochemical validation to provide insights into the mechanisms of oxidative inactivation of GLRX.
- Elizabeth M. Corteselli
- , Mona Sharafi
- & Yvonne M. W. Janssen-Heininger
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Article
| Open AccessA cytochrome P450 CYP87A4 imparts sterol side-chain cleavage in digoxin biosynthesis
Digoxin is a heart medicine extracted from plants, but how plants synthesize it is largely unknown. Here Carroll et al. identify a novel enzyme for digoxin biosynthesis, paving the way to produce digoxin and other structurally similar drugs in microbes.
- Emily Carroll
- , Baradwaj Ravi Gopal
- & Zhen Q. Wang
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Article
| Open AccessA flavin-monooxygenase catalyzing oxepinone formation and the complete biosynthesis of vibralactone
Vibralactone is a strong lipase inhibitor and a bicyclic β-lactone containing an oxepinone ring, whose biosynthetic construction was unknown. Here the authors identify an oxepinone-building flavin monooxygenase VibO that is involved in the biosynthesis of vibralactone, and determine its X-ray crystal structure.
- Ke-Na Feng
- , Yue Zhang
- & Ying Zeng
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Article
| Open AccessA 2.2 Å cryoEM structure of a quinol-dependent NO Reductase shows close similarity to respiratory oxidases
Quinol-dependent nitric oxide reductases, unique to bacteria, are considered members of respiratory heme copper oxidases. A 2.2 Å cryoEM structure of qNOR is reported shedding light on key aspects of enzyme mechanism including quinol binding and pathways for electron, substrate, and proton transport.
- Alex J. Flynn
- , Svetlana V. Antonyuk
- & S. Samar Hasnain
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Article
| Open AccessStructural consequences of turnover-induced homocitrate loss in nitrogenase
Biological nitrogen fixation is achieved by nitrogenase, but the mechanism remains enigmatic. Here, the authors report high resolution single particle cryoEM structures of homocitrate-compromised MoFe-proteins and unveil a new binding partner.
- Rebeccah A. Warmack
- , Ailiena O. Maggiolo
- & Douglas C. Rees
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Article
| Open AccessEvolution of enzyme functionality in the flavin-containing monooxygenases
Detoxification enzymes are crucial for the survival of animals in new environments. Here, the authors study the molecular mechanism behind the catalytic diversification of a major family of tetrapod detoxification enzymes—the FMOs—during evolution.
- Gautier Bailleul
- , Guang Yang
- & Maria Laura Mascotti
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Article
| Open AccessVisible light-exposed lignin facilitates cellulose solubilization by lytic polysaccharide monooxygenases
Degradation of plant biomass, comprised of cellulose and polyaromatic lignin, is promoted by light. Here, the authors show that light promotes lignin-catalyzed generation of hydrogen peroxide, which is used by redox enzymes to degrade cellulose.
- Eirik G. Kommedal
- , Camilla F. Angeltveit
- & Vincent G. H. Eijsink
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Article
| Open AccessA genome-wide CRISPR-Cas9 knockout screen identifies FSP1 as the warfarin-resistant vitamin K reductase
The authors develop a Vitamin K-dependent apoptotic reporter cell line for large-scale screening of enzymes associated with Vitamin K-dependent carboxylation and identify ferroptosis suppressor protein 1 (FSP1) as responsible for warfarin-resistant vitamin K reduction.
- Da-Yun Jin
- , Xuejie Chen
- & Jian-Ke Tie
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Article
| Open AccessDiscovery and biosynthesis of karnamicins as angiotensin converting enzyme inhibitors
Treatment of hypertension entails use of angiotensin-converting enzyme inhibitors. Here, the authors show a series of karnamicins with significant inhibitory activity and identify two unusual flavoprotein hydroxylases involved in the assembly of the fully-substituted hydroxypyridine core of karnamicins.
- Zhiyin Yu
- , Jian-Ping Huang
- & Sheng-Xiong Huang
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Article
| Open AccessStructure- and computational-aided engineering of an oxidase to produce isoeugenol from a lignin-derived compound
Lignin can be depolymerized into 4-alkylphenols by chemical means. Here the authors show a three-step computational-assisted enzyme engineering process to generate a biocatalyst for the conversion of lignin-derived 4-n-propylguaiacol into isoeugenol, a valuable compound.
- Yiming Guo
- , Laura Alvigini
- & Marco W. Fraaije
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Article
| Open AccessEndothelial alpha globin is a nitrite reductase
In mammals, hypoxia causes dilation of small arteries for increased metabolic demand. Keller et al used novel transgenic mice to show alpha hemoglobin in endothelium, once thought only in red blood cells, can regulate hypoxic-mediated dilation.
- T. C. Stevenson Keller IV
- , Christophe Lechauve
- & Brant E. Isakson
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Article
| Open AccessStructure of the core human NADPH oxidase NOX2
NADPH oxidase NOX2 produces superoxide, a reactive oxygen species essential in innate immunity. Here, the authors reveal the structure of the NOX2 core, rationalize disease-causing mutations, and suggest avenues for selective NOX2 pharmacological modulation.
- Sigrid Noreng
- , Naruhisa Ota
- & James T. Koerber
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Article
| Open AccessBiochemical and structural insights of multifunctional flavin-dependent monooxygenase FlsO1-catalyzed unexpected xanthone formation
The biosynthesis of xanthones has not been well documented. Here, the authors report that monooxygenase FlsO1 catalyzes three successive oxidations – hydroxylation, epoxidation and Baeyer–Villiger oxidation—to form the xanthone scaffold in actinomycetes.
- Chunfang Yang
- , Liping Zhang
- & Changsheng Zhang
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Article
| Open AccessResistance to the isocitrate dehydrogenase 1 mutant inhibitor ivosidenib can be overcome by alternative dimer-interface binding inhibitors
The development of IDH variant inhibitors is a breakthrough as it is the first time metabolism has been successfully targeted by small molecule drugs in cancer. Here the authors report studies on resistance to the pioneer drug ivosidenib leading to identification of inhibitors retaining activity.
- Raphael Reinbold
- , Ingvild C. Hvinden
- & Christopher J. Schofield
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Article
| Open AccessCyclic 5-membered disulfides are not selective substrates of thioredoxin reductase, but are opened nonspecifically
Cyclic five-membered disulfides (1,2-dithiolanes) have been reported either as nonspecific redox motifs, or as highly specific cellular probes for thioredoxin reductase (TrxR). Here the authors show that 1,2-dithiolane probes are nonspecifically reduced by a range of thiol reductants and are not sensitive to TrxR modulation, thus they are unsuitable as cellular probes for TrxR.
- Jan G. Felber
- , Lena Poczka
- & Oliver Thorn-Seshold
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Article
| Open AccessBerberine bridge enzyme-like oxidase-catalysed double bond isomerization acts as the pathway switch in cytochalasin synthesis
Cytochalasans are a large family of fungal polyketide-nonribosomal peptide hybrid natural products that exhibit important pharmaceutical activities, but the mechanism of conversion of the monocytochalasans to the polycyclic, fused analogues is unclear. Here the authors reconstitute the core backbone of the cytochalasin family and describe an oxidase that catalyzes an unusual double-bond isomerization reaction.
- Jin-Mei Zhang
- , Xuan Liu
- & Yi Zou
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Article
| Open AccessReductive inactivation of the hemiaminal pharmacophore for resistance against tetrahydroisoquinoline antibiotics
Antibiotic-producing organisms need to co-evolve self-protection mechanisms to avoid any damage to themselves caused by the antibiotic pharmacophore (the reactive part of the compound). In this study, the authors report a self-defense strategy in naphthyridinomycin (NDM)-producing Streptomyces lusitanus, that comprises reductive inactivation of the hemiaminal pharmacophore by short-chain dehydrogenases/reductases (SDRs) NapW and HomW.
- Wan-Hong Wen
- , Yue Zhang
- & Gong-Li Tang
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Article
| Open Access2-Oxoglutarate derivatives can selectively enhance or inhibit the activity of human oxygenases
The human 2-oxoglutarate (2OG) oxygenases FIH and AspH are relevant drug targets. Here, the authors show that synthetic and naturally occurring 2OG derivatives can selectively modulate FIH and AspH activities, suggesting that these compounds may serve as a basis to develop 2OG oxygenase-targeting probes and drugs.
- Yu Nakashima
- , Lennart Brewitz
- & Christopher J. Schofield
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Article
| Open AccessFlavin-dependent halogenases catalyze enantioselective olefin halocyclization
Catalytic enantioselective halocyclization of alkenes is an important bond forming tool and a key step in natural product biosynthesis, but so far no examples of the enzymatic counterpart of this reaction on simple achiral olefins have been reported. Here, the authors describe examples of engineered flavin-dependent halogenases that catalyze halolactonization of olefins with high enantioselectivity and near-native catalytic activity.
- Dibyendu Mondal
- , Brian F. Fisher
- & Jared C. Lewis
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Article
| Open AccessDiscovery of fungal oligosaccharide-oxidising flavo-enzymes with previously unknown substrates, redox-activity profiles and interplay with LPMOs
Microbial oxidoreductases are key in biomass breakdown. Here, the authors expand the specificity and redox scope within fungal auxiliary activity 7 family (AA7) enzymes and show that AA7 oligosaccharide dehydrogenases can directly fuel cellulose degradation by lytic polysaccharide monooxygenases.
- Majid Haddad Momeni
- , Folmer Fredslund
- & Maher Abou Hachem
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Article
| Open AccessA [6+4]-cycloaddition adduct is the biosynthetic intermediate in streptoseomycin biosynthesis
Streptoseomycin is a potent antibiotic that contains a pentacyclic 5/14/10/6/6 ring system. Here, the authors report the enzymatic and non-enzymatic steps of the downstream modification of streptoseomycin biosynthesis and show a [6 + 4]-cycloaddition adduct as an unexpected biosynthetic intermediate.
- Kai Biao Wang
- , Wen Wang
- & Hui Ming Ge
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Article
| Open AccessDirect detection of coupled proton and electron transfers in human manganese superoxide dismutase
Human manganese superoxide dismutase (MnSOD) is an oxidoreductase that uses concerted proton and electron transfers to reduce the levels of superoxide radicals in mitochondria, but mechanistic insights into this process are limited. Here, the authors report neutron crystal structures of Mn3+SOD and Mn2+SOD, revealing changes in the protonation states of key residues in the enzyme active site during the redox cycle.
- Jahaun Azadmanesh
- , William E. Lutz
- & Gloria E. O. Borgstahl
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Article
| Open AccessStructural characterization of the microbial enzyme urocanate reductase mediating imidazole propionate production
Imidazole propionate (ImP) produced by gut microbiota has been associated with type 2 diabetes. Here, the authors present crystal structures of the ImP biosynthesis enzyme urocanate reductase in four different states, providing molecular insights into its catalytic mechanism.
- Raminta Venskutonytė
- , Ara Koh
- & Karin Lindkvist-Petersson
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Article
| Open AccessThe biosynthetic pathway of potato solanidanes diverged from that of spirosolanes due to evolution of a dioxygenase
One goal of potato breeding is to reduce the accumulation of toxic solanidane glycoalkaloids. Here the authors show that potato DPS, a 2-oxoglutarate dependent dioxygenase, catalyzes ring rearrangement of a biosynthetic precursor to differentiate solanidanes from spirosolanes that are found in other solanaceous plants.
- Ryota Akiyama
- , Bunta Watanabe
- & Masaharu Mizutani
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Article
| Open AccessA rotary mechanism for allostery in bacterial hybrid malic enzymes
Bacterial malic enzymes (ME) transform malate to pyruvate. One group, hybrid ME enzymes, are regulated by acetyl-CoA, linking the enzyme activity to the metabolic state of the cell. Structures of a representative hybrid ME MaeB reveal large conformational rearrangements that provide insight into the mechanism of allosteric inhibition by acetyl-CoA.
- Christopher John Harding
- , Ian Thomas Cadby
- & Andrew Lee Lovering
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Article
| Open AccessThe lytic polysaccharide monooxygenase CbpD promotes Pseudomonas aeruginosa virulence in systemic infection
The Pseudomonas aeruginosa lytic polysaccharide monooxygenase CbpD, prevalent in clinical isolates, has been proposed to act as a virulence factor. Here, the authors combine structural work, in silico simulations, enzymatic activity and in vitro and in vivo experiments to further delineate the role of CbpD and show that its deletion renders P. aeruginosa unable to establish a lethal systemic infection, leading to enhanced bacterial clearance in a mouse model of infection.
- Fatemeh Askarian
- , Satoshi Uchiyama
- & Gustav Vaaje-Kolstad
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Article
| Open AccessFunctional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor
An important type of post-translational protein modification is the conversion of peptidyl amino acid into enzyme cofactor. Here, the authors report functional and structural characterization of a flavoprotein monooxygenase essential for biosynthesis of cysteine tryptophylquinone (CTQ) cofactor.
- Toshinori Oozeki
- , Tadashi Nakai
- & Toshihide Okajima
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Article
| Open AccessStructures of human dual oxidase 1 complex in low-calcium and high-calcium states
Dual oxidases (DUOXs), assembled from the catalytic DUOX and the auxiliary DUOXA subunits, produce hydrogen peroxide by transferring electrons from intracellular NADPH to extracellular oxygen in a calcium-activated manner. Here authors report the cryo-EM structures of human DUOX1-DUOXA1 complex in both high-calcium and low-calcium states.
- Jing-Xiang Wu
- , Rui Liu
- & Lei Chen
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Article
| Open AccessKinetic insights into the peroxygenase activity of cellulose-active lytic polysaccharide monooxygenases (LPMOs)
Lytic polysaccharide monooxygenases (LPMOs) catalyze the hydroxylation of glycosidic bonds in polysaccharides, but the catalytic properties of these monocopper enzymes remain poorly characterized. Here authors employ competition between reference enzymes and LPMOs for the H2O2 co-substrate to kinetically characterize LPMO-catalyzed cellulose oxidation.
- Riin Kont
- , Bastien Bissaro
- & Priit Väljamäe
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Article
| Open AccessStructure of inhibitor-bound mammalian complex I
The respiratory complex I (NADH:ubiquinone oxidoreductase) is a large redox-driven proton pump that initiates respiration in mitochondria. Here, the authors present the 3.0 Å cryo-EM structure of complex I from mouse heart mitochondria with the ubiquinone-analogue inhibitor piericidin A bound in the active site and with kinetic measurements and MD simulations they further show that this inhibitor acts competitively against the native ubiquinone-10 substrate.
- Hannah R. Bridges
- , Justin G. Fedor
- & Judy Hirst
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Article
| Open AccessUncovering the cytochrome P450-catalyzed methylenedioxy bridge formation in streptovaricins biosynthesis
Streptovaricin C is an antibiotic containing a methylenedioxy bridge (MDB) moiety essential for its activity. Here, the authors show that a P450 monooxygenase StvP2 catalyses MDB formation, report its crystal structure in complex with the substrate, and elucidate mechanistic details of MDB formation.
- Guo Sun
- , Chaoqun Hu
- & Yuhui Sun
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Article
| Open AccessATP-dependent hydroxylation of an unactivated primary carbon with water
Monooxygenases catalyse the hydroxylation of C-H bonds using oxygen as a co-substrate, which, in turn, is unavailable for anaerobic bacteria. Here, the authors report a three-step reaction cascade involving two hydroxylases and one dehydratase which hydroxylate the C26 methyl group of cholesterol with water as a co-substrate.
- Christian Jacoby
- , Sascha Ferlaino
- & Matthias Boll
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Article
| Open AccessPhotoinduced monooxygenation involving NAD(P)H-FAD sequential single-electron transfer
The number of usable light-responsive enzymes is limited, despite the potential biotechnological applications. Here, the authors report a flavoprotein monooxygenase which is controllable by blue light illumination, and propose a mechanism involving protein-mediated radical photoreduction of FAD via a semiquinone intermediate.
- Simon Ernst
- , Stefano Rovida
- & Steffen L. Drees
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Article
| Open AccessNitric oxide orchestrates metabolic rewiring in M1 macrophages by targeting aconitase 2 and pyruvate dehydrogenase
Production of inflammatory mediators by M1-polarized macrophages is thought to rely on suppression of mitochondrial metabolism in favor of glycolysis. Refining this concept, here the authors define metabolic targets of nitric oxide as responsible for the mitochondrial rewiring resulting from polarization.
- Erika M. Palmieri
- , Marieli Gonzalez-Cotto
- & Daniel W. McVicar
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Article
| Open AccessAssessing electron transfer reactions and catalysis in multicopper oxidases with operando X-ray absorption spectroscopy
Understanding enzyme active sites can elucidate fundamental enzymatic reaction pathways and inform designs for synthetic catalysts. Here, authors employ operando X-ray absorption spectroelectrochemistry to assess copper ions in bilirubin oxidase during oxygen reduction electrocatalysis.
- Lucyano J. A. Macedo
- , Ayaz Hassan
- & Frank N. Crespilho
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Article
| Open AccessCytosolic ROS production by NADPH oxidase 2 regulates muscle glucose uptake during exercise
Reactive oxygen species (ROS) stimulate GLUT4-mediated glucose transport following contraction of isolated muscle, but it is not clear if this occurs in vivo. Here, the authors show in human volunteers that exercise induces ROS increase in muscle and, using loss of-function animal models, they demonstrate that NOX2 is a major ROS source required to stimulate glucose uptake during exercise.
- Carlos Henríquez-Olguin
- , Jonas R. Knudsen
- & Thomas E. Jensen
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Article
| Open AccessA mechanism to prevent production of reactive oxygen species by Escherichia coli respiratory complex I
Respiratory complex I plays a central role in cellular energy metabolism coupling NADH oxidation to proton translocation. Here, the authors report the structure of the electron input part of Aquifex aeolicus complex I at up to 1.8 Å resolution with bound substrates in the reduced and oxidized states.
- Marius Schulte
- , Klaudia Frick
- & Thorsten Friedrich
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Article
| Open AccessAminoacyl sulfonamide assembly in SB-203208 biosynthesis
Sulfonamide is in many important drugs yet is rare in nature and little is known about the synthesis of sulfonamide containing antibiotics. Here, the authors report on a detailed analysis of the biosynthesis machineries of the aminoacyl sulfonamide antibiotics.
- Zhijuan Hu
- , Takayoshi Awakawa
- & Ikuro Abe
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Article
| Open AccessMolecular basis of maintaining an oxidizing environment under anaerobiosis by soluble fumarate reductase
Soluble fumarate reductases are essential for eukaryotic cell survival under anaerobic conditions but their mechanism is not fully understood. Here, the authors present structural and enzymatic analyses of yeast fumarate reductase Osm1, elucidating the molecular basis of maintaining redox balance during anaerobiosis.
- Sunghwan Kim
- , Chang Min Kim
- & Hyun Ho Park
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Article
| Open AccessStructural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine inhibition
The human enzyme MTHFR links the folate and methionine cycles, which are essential for the biosynthesis of nucleotides and proteins. Here, the authors present the crystal structure and biochemical analysis of human MTHFR, providing molecular insights into its function and regulation in higher eukaryotes.
- D. Sean Froese
- , Jolanta Kopec
- & Wyatt W. Yue
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Article
| Open AccessStructural basis for energy transduction by respiratory alternative complex III
Some prokaryotes use alternative respiratory chain complexes, such as the alternative complex III (ACIII), to generate energy. Here authors provide the cryoEM structure of ACIII from Rhodothermus marinus which shows the arrangement of cofactors and provides insights into the mechanism for energy transduction.
- Joana S. Sousa
- , Filipa Calisto
- & Manuela M. Pereira
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Article
| Open AccessCrystal structure of an assembly intermediate of respiratory Complex II
The mechanism for covalent flavinylation of flavoenzymes is still unclear. Here, the authors propose a mechanism based on the crystal structure of a flavinylation assembly intermediate of the E. coli respiratory Complex II comprising the E. coli FrdA subunit bound to covalent FAD and crosslinked with its assembly factor SdhE.
- Pankaj Sharma
- , Elena Maklashina
- & T. M. Iverson