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| Open AccessNucleotide binding halts diffusion of the eukaryotic replicative helicase during activation
Here the authors examine fully reconstituted and activated CMG helicases at the single-molecule level. They observe unidirectional translocation by CMG in ATP and diffusive motion by CMG without ATP, where the latter can be halted by nucleotide binding.
- Daniel Ramírez Montero
- , Humberto Sánchez
- & Nynke H. Dekker
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Article
| Open AccessExtended DNA threading through a dual-engine motor module of the activating signal co-integrator 1 complex
ASCC3 is a multi-functional helicase that contains two consecutive Ski2-like helicase units. Here, the authors show that ASCC3 can unwind DNA by threading one strand of a substrate duplex through both helicase units, supported by the TRIP4 protein.
- Junqiao Jia
- , Tarek Hilal
- & Markus C. Wahl
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Article
| Open AccessMolecular mechanism on forcible ejection of ATPase inhibitory factor 1 from mitochondrial ATP synthase
IF1 is a natural inhibitor of mitochondrial FoF1-ATP synthase, which blocks catalysis and rotation of the F1 motor. Here, the authors show the rotational-direction-dependence of activation from IF1 inhibition, with IF1 being readily dissociated when F1 rotates to the clockwise direction.
- Ryohei Kobayashi
- , Hiroshi Ueno
- & Hiroyuki Noji
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Article
| Open AccessUnnatural activities and mechanistic insights of cytochrome P450 PikC gained from site-specific mutagenesis by non-canonical amino acids
The 20 proteinogenic amino acids cannot always satisfy the requirements of protein engineering. Here, the authors practice semi-rational non-canonical amino acid mutagenesis for a cytochrome P450 enzyme, leading to unnatural activities and significant mechanistic insights.
- Yunjun Pan
- , Guobang Li
- & Shengying Li
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Article
| Open AccessC–N bond formation by a polyketide synthase
Polyketide synthases (PKSs) usually form C–C bonds in natural products biosynthesis. Here, the authors present cryo-EM structures of a PKS in complex with products, which provides insight into the mechanism of the unexpected C–N bond formation.
- Jialiang Wang
- , Xiaojie Wang
- & Jingdan Liang
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Article
| Open AccessrRNA methylation by Spb1 regulates the GTPase activity of Nog2 during 60S ribosomal subunit assembly
Regulation of 60S biogenesis remains poorly understood. Using cryo-EM, the authors show that failure of Spb1 to methylate the A-loop nucleotide G2922 prematurely activates the GTPase Nog2, suggesting that Spb1 and Nog2 form a kinetic checkpoint during ribosome maturation.
- Kamil Sekulski
- , Victor Emmanuel Cruz
- & Jan P. Erzberger
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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 AccessStructural remodelling of the carbon–phosphorus lyase machinery by a dual ABC ATPase
Here, authors analyse the structural organisation of the large carbon-phosphorus lyase enzyme from bacteria using electron microscopy and discover that it contains two ATP-binding cassette dimers of PhnK and PhnL and opens upon ATP hydrolysis.
- Søren K. Amstrup
- , Sui Ching Ong
- & Ditlev E. Brodersen
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Article
| Open AccessSmall molecule inhibitors of 15-PGDH exploit a physiologic induced-fit closing system
Inhibition of 15-prostaglandin dehydrogenase (15-PGDH) is a promising therapeutic target for regenerative medicine. We report the structure of 15-PGDH in complex with two different inhibitors. Unexpectedly, access to the binding pocket is regulated by a dynamic “lid” of the enzyme.
- Wei Huang
- , Hongyun Li
- & Derek J. Taylor
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Article
| Open AccessInsights into azalomycin F assembly-line contribute to evolution-guided polyketide synthase engineering and identification of intermodular recognition
Intermodular recognition in polyketide synthase (PKS) is a key prerequisite for catalysis and assembly-line engineering. Here, the authors present a specific domain interaction between modules and an evolution-oriented strategy for PKS engineering derived from the enoylreduction module of azalomycin F.
- Guifa Zhai
- , Yan Zhu
- & Yuhui Sun
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Article
| Open AccessAn engineered variant of MECR reductase reveals indispensability of long-chain acyl-ACPs for mitochondrial respiration
Mitochondrial fatty acid synthesis (mtFAS) generates the precursor for lipoic acid synthesis, but the role of longer fatty acid products has remained unclear. Here, the authors generated an engineered variant of human 2E-enoyl-ACP reductase (MECR) of mtFAS to study the role of long chain fatty acids.
- M. Tanvir Rahman
- , M. Kristian Koski
- & Kaija J. Autio
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Article
| Open AccessEmergence of a proton exchange-based isomerization and lactonization mechanism in the plant coumarin synthase COSY
Plant biosynthetic enzymes rapidly evolve to catalyze specialized reactions. Here, the authors present the crystal structure and mechanism of COSY, the enzyme involved in coumarin biosynthesis of the BAHD-acyltransferase family that catalyzes an intramolecular acyl transfer reaction through a proton exchange mechanism.
- Colin Y. Kim
- , Andrew J. Mitchell
- & Jing-Ke Weng
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Article
| Open AccessStructural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization
Biological processes can be regulated via oligomerization of macromolecules into high-order symmetric structures. Here, authors reported high-order structure of RNA polymerase and its role in regulation of gene expression in pathogenic bacterium.
- Zakia Morichaud
- , Stefano Trapani
- & Konstantin Brodolin
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Article
| Open AccessStructural analysis of an anthrol reductase inspires enantioselective synthesis of enantiopure hydroxycycloketones and β-halohydrins
Asymmetric reduction of prochiral ketones is challenging. Here, the authors identify and solve the structure of anthrol reductase CbAR, whose variant H162F can convert 1,3-cyclodiketones and α-haloacetophenones to the corresponding chiral alcohols.
- Xiaodong Hou
- , Huibin Xu
- & Yijian Rao
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Article
| Open AccessInsights into the mechanism of phospholipid hydrolysis by plant non-specific phospholipase C
Non-specific phospholipase C (NPC) is a plant-specific phospholipid-hydrolyzing enzyme. Here, the authors define the molecular basis of how Arabidopsis NPC works, and provide new mechanistic insights into the members of phospholipase family.
- Ruyi Fan
- , Fen Zhao
- & Zhu Liu
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Article
| Open AccessThe unifying catalytic mechanism of the RING-between-RING E3 ubiquitin ligase family
RBR E3 ubiquitin ligases utilise a 2-step catalytic mechanism previously defined for only few of the RBR family members. Here, the authors examine the poorly studied RBRs HOIL-1 and RNF216 to define general principles of RBR catalysis and regulation and identify specific functional differences.
- Xiangyi S. Wang
- , Thomas R. Cotton
- & Bernhard C. Lechtenberg
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Article
| Open AccessA conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1
SARM1 is a key player in axon degeneration. Here, the authors generate a nanobody, which specifically recognizes the NMN-bound state of SARM1 and helps resolve the SARM1 structure in an intermediate state of activation.
- Yun Nan Hou
- , Yang Cai
- & Yong Juan Zhao
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Article
| Open AccessThe UFM1 system regulates ER-phagy through the ufmylation of CYB5R3
The UFM1 system, a ubiquitin-like conjugation system is crucial for endoplasmic reticulum (ER) homeostasis. Here, authors found that CYB5R3 is covalently conjugated with UFM1, which becomes a signal for ER-phagy, a selective autophagy of ER.
- Ryosuke Ishimura
- , Afnan H. El-Gowily
- & Masaaki Komatsu
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Article
| Open AccessMechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing
In this work, Blair and co-authors used cryo-EM and in vitro assays to show that human Ligase 1 (Lig1) and Flap Endonuclease 1 (FEN1) form a toolbelt with the sliding clamp PCNA coordinating the sealing of Okazaki fragments.
- Kerry Blair
- , Muhammad Tehseen
- & Alfredo De Biasio
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Article
| Open AccessRational enzyme design for enabling biocatalytic Baldwin cyclization and asymmetric synthesis of chiral heterocycles
Chiral heterocyclic compounds are privileged structures in medicinal chemistry. Here, the authors report an in silico strategy for the enzymatic synthesis of pharmaceutically significant chiral N- and O-heterocycles via Baldwin cyclization of hydroxy- and amino-substituted epoxides and oxetanes using epoxide hydrolase mutants.
- Jun-Kuan Li
- , Ge Qu
- & Zhoutong Sun
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Article
| Open AccessStructural insights into p300 regulation and acetylation-dependent genome organisation
Here the authors use structural analyses to show that an intrinsically disordered transcription activation domain in the oncogene BRD4-NUT binds to and activates p300. This in-turn drives formation of higher-order, acetylation-dependent chromatin condensates.
- Ziad Ibrahim
- , Tao Wang
- & Daniel Panne
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Article
| Open AccessAllosteric rescue of catalytically impaired ATP phosphoribosyltransferase variants links protein dynamics to active-site electrostatic preorganisation
ATPPRT is a multi-protein allosteric enzyme where the regulatory protein HisZ enhances catalysis by HisGs. Here, the authors report catalytically impaired active site mutants of HisGs that are allosterically rescued by HisZ despite the HisZ:HisGs interface lying ~20 Å away from the active site.
- Gemma Fisher
- , Marina Corbella
- & Rafael G. da Silva
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Article
| Open AccessDivergent self-association properties of paralogous proteins TRIM2 and TRIM3 regulate their E3 ligase activity
TRIM E3 ligases are key regulators of numerous cellular functions, yet their mechanism of action remains poorly understood. Here, the authors show that paralogous TRIM2 and TRIM3 adopt different self-association and activity profiles but interact in cells to modulate their respective activities.
- Diego Esposito
- , Jane Dudley-Fraser
- & Katrin Rittinger
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Article
| Open AccessThe role of auxiliary domains in modulating CHD4 activity suggests mechanistic commonality between enzyme families
CHD4 is an essential chromatin remodelling enzyme in complex organisms. Here the authors demonstrate that CHD4 activity is regulated both positively and negatively by distinct auxiliary domains, revealing parallels with other chromatin remodellers.
- Yichen Zhong
- , Hakimeh Moghaddas Sani
- & Joel P. Mackay
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Article
| Open AccessA quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex
Based on thorough single-molecule analysis of off-target binding by the CRISPR-Cas surveillance complex Cascade, a mechanism-based model is presented that quantitatively describes the recognition dynamics of mutated targets by this protein.
- Marius Rutkauskas
- , Inga Songailiene
- & Ralf Seidel
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Article
| Open AccessMolecular basis for glycan recognition and reaction priming of eukaryotic oligosaccharyltransferase
Oligosaccharyltransferase (OST), the central enzyme in N-glycosylation, modifies acceptor proteins by attaching a complex glycan. Cryo-EM structures of OST in distinct states, reveal the molecular basis of substrate recognition and catalysis.
- Ana S. Ramírez
- , Mario de Capitani
- & Kaspar P. Locher
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Article
| Open AccessA marine sponge-derived lectin reveals hidden pathway for thrombopoietin receptor activation
The mode of cytokine receptor activation is diverse. Here, the authors find that the marine-sponge derived lectin ThC, a bivalent sugar binding protein, activates human cytokine receptor MPL. This mode of action resembles the pathogenic activation of MPL by mutant molecular chaperon calreticulin in hematologic malignancies.
- Hiromi Watari
- , Hiromu Kageyama
- & Ryuichi Sakai
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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 AccessStructure of the human heparan sulfate polymerase complex EXT1-EXT2
Heparan sulfates are long and complex polysaccharides that mediate a large number of biological processes at the cell surface. Here, the authors provide structural and functional insights into the human EXT1-EXT2 complex that carries out the polymerization of heparan sulfate chains.
- Francisco Leisico
- , Juneina Omeiri
- & Rebekka Wild
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Article
| Open AccessStructural and functional asymmetry of RING trimerization controls priming and extension events in TRIM5α autoubiquitylation
TRIM5α is an E3 ligase that inhibits retroviral replication. Here, the authors delineate the biochemical mechanism that accelerates N terminal autoubiquitylation of TRIM5α upon its association with the retroviral capsid and, thus, enables recognition of an infection-associated molecular pattern.
- Frank Herkules
- , Corey H. Yu
- & Dmitri N. Ivanov
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Article
| Open AccessDNA mechanical flexibility controls DNA potential to activate cGAS-mediated immune surveillance
DNA is well-documented to stimulate immune response. Here the authors show that the activation of cyclic GMP-AMP synthase (cGAS) depends on DNA mechanical flexibility, itself determined by DNA-sequence, damage and length.
- Lina Wang
- , Siru Li
- & Qingkai Yang
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Article
| Open AccessStructure of the catalytically active APOBEC3G bound to a DNA oligonucleotide inhibitor reveals tetrahedral geometry of the transition state
Here, the enzymatic activity of APOBEC3G resulting in conversion of 2′-deoxy-zebularine into a hydration product allowed the authors to capture the transition state, which provides a blueprint for designing a new class of transition state-mimicking inhibitors for this class of enzyme.
- Atanu Maiti
- , Adam K. Hedger
- & Hiroshi Matsuo
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Article
| Open AccessMechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics
Here, the authors investigate the maturation of Okazaki fragments with human proteins and reveal that initiator RNA removal occurs non-processively and slowly suggesting that additional pathways might exist to accelerate RNA removal in cells.
- Vlad-Stefan Raducanu
- , Muhammad Tehseen
- & Samir M. Hamdan
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Article
| Open AccessCryo-EM structures reveal the activation and substrate recognition mechanism of human enteropeptidase
Dysfunction of enteropeptidase (EP) can result in pancreatitis, a life-threatening disease. Yang et al. report a series of EP structures representing the EP catalytic cycle and propose a mechanistic model of enzyme function.
- Xiaoli Yang
- , Zhanyu Ding
- & Haojie Huang
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Article
| Open AccessCK2-mediated phosphorylation of SUZ12 promotes PRC2 function by stabilizing enzyme active site
Here the authors identify SUZ12 as a cellular substate of casein kinase 2 (CK2), and show this phosphorylation changes the active site structure of Polycomb repressive complex 2 (PRC2) and promotes PRC2 function in cell identity maintenance during stem cell differentiation.
- Lihu Gong
- , Xiuli Liu
- & Xin Liu
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Article
| Open AccessStructural basis for sequence-independent substrate selection by eukaryotic wobble base tRNA deaminase ADAT2/3
The deamination of all adenosines in the wobble base position is essential to empower the individual tRNA to decode several codons. Here, the authors present the cryo-EM structure of the tRNA-bound ADAT2/3 deaminase, revealing how the geometry-specific enzyme acquiesces sequence-divergent tRNAs to its active site.
- Luciano G. Dolce
- , Aubree A. Zimmer
- & Eva Kowalinski
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Article
| Open AccessEndowing homodimeric carbamoyltransferase GdmN with iterative functions through structural characterization and mechanistic studies
Carbamoyltransferases are a class of enzymes catalyzing carbamoylation of primary and secondary metabolites. Here, the authors show the molecular structure of the ansamycin-modifying carbamoyltransferase GdmN and repurpose the enzyme for the iterative carbamoylation of ansamitocins.
- Jianhua Wei
- , Xuan Zhang
- & Linquan Bai
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Article
| Open AccessStructural dynamics of DNA strand break sensing by PARP-1 at a single-molecule level
Sm FRET studies of PARP-1 recognition of DNA lesion show that binding of F1F2 fragment results in a kinked DNA, while binding of F2 fragment alone yields an intermediate state. This dynamic equilibrium of states is influenced by inhibitors.
- Anna Sefer
- , Eleni Kallis
- & Jens Michaelis
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Article
| Open AccessUnexpected assembly machinery for 4(3H)-quinazolinone scaffold synthesis
4(3H)-quinazolinone is the core scaffold in more than 200 natural alkaloids and numerous drugs. Here, the authors show an alternative assembly machinery for 4(3H)-quinazolinone mainly includes a two-module NRPS catalysing tripeptide formation, unusual N-3 original sources and an α-KGD catalysing the C-N bond oxidative cleavage of a tripeptide to form a dipeptide.
- Xi-Wei Chen
- , Li Rao
- & Yi Zou
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Article
| Open AccessFunctional selectivity of insulin receptor revealed by aptamer-trapped receptor structures
DNA aptamers can activate insulin receptor as selective agonists or positive allosteric modulators. Here, authors determine structures of the insulin receptor bound to aptamers and provide a basis for the selective activation or allosteric regulation of insulin receptor.
- Junhong Kim
- , Na-Oh Yunn
- & Yunje Cho
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Article
| Open AccessMolecular insights into DNA recognition and methylation by non-canonical type I restriction-modification systems
Type I R-M systems help establish the prokaryotic DNA methylation landscape and provide protection against invasive DNA. Here, the authors report on detailed structural and molecular mechanisms of a non-canonical type I R-M methyltransferase.
- Jingpeng Zhu
- , Yina Gao
- & Pu Gao
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Article
| Open AccessA fungal NRPS-PKS enzyme catalyses the formation of the flavonoid naringenin
Biosynthesis of the flavonoid naringenin in plants and bacteria is commonly catalysed by a type III polyketide synthase (PKS) using one p-coumaroyl-CoA and three malonyl-CoA molecules as substrates. Here, the authors report a fungal non-ribosomal peptide synthetase PKS hybrid FnsA catalysing the formation of naringenin.
- Hongjiao Zhang
- , Zixin Li
- & Wen-Bing Yin
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Article
| Open AccessPurification and structural characterization of the Na+-translocating ferredoxin: NAD+ reductase (Rnf) complex of Clostridium tetanomorphum
Rnf reversibly reduces NAD+ by ferredoxin coupled with Na+/H+ pumping for microbial ion gradient or reduced ferredoxin formation. Here, a cryo-EM structure provides information about the participating iron and flavin cofactors, the electron transfer route and the putative Na+ passage.
- Stella Vitt
- , Simone Prinz
- & Wolfgang Buckel
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Article
| Open AccessEngineered helicase replaces thermocycler in DNA amplification while retaining desired PCR characteristics
PCR is an essential method for the amplification and manipulation of nucleic acids, but the requirement for a thermocycler limits access. Here, authors engineer a helicase to replace the heating step of PCR with enzymatic unwinding, allowing the isothermal amplification of fragments up to 6 kb.
- Momčilo Gavrilov
- , Joshua Y. C. Yang
- & Taekjip Ha
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Article
| Open AccessThe retaining β-Kdo glycosyltransferase WbbB uses a double-displacement mechanism with an intermediate adduct rearrangement step
WbbB is a structurally unusual retaining glycosyltransferase. Here, the authors show that WbbB forms an Asp232-Kdo adduct prior to transfer to the saccharide acceptor. Therefore, unlike any previously studied glycosyltransferase, WbbB uses the double-displacement mechanism first proposed in 1953.
- Taylor J. B. Forrester
- , Olga G. Ovchinnikova
- & Matthew S. Kimber
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Article
| Open AccessStructural insight into the ligand binding mechanism of aryl hydrocarbon receptor
The aryl hydrocarbon receptor (AHR) plays important roles in regulating xenobiotic metabolism, cellular differentiation, and immunity. Here, the authors present multiple crystal structures of the Drosophila AHR PAS-B domain, providing insights into the mechanism of AHR ligand binding
- Shuyan Dai
- , Lingzhi Qu
- & Yongheng Chen
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Article
| Open AccessCryoEM structural exploration of catalytically active enzyme pyruvate carboxylase
Pyruvate Carboxylase is a multifunctional enzyme that follows a multi-pathway reaction. Here, the authors, using cryoEM and classification techniques, reveal several catalytic states at reaction sites, the interplay between them, and their relationship with motions in the tetrameric organization.
- Jorge Pedro López-Alonso
- , Melisa Lázaro
- & Mikel Valle
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Article
| Open AccessIon transfer mechanisms in Mrp-type antiporters from high resolution cryoEM and molecular dynamics simulations
Many bacteria employ unique membrane transport systems to survive harsh environmental conditions. Here, authors determined the structure of a cation/proton antiporter at high resolution and studied its mechanism by computer simulations.
- Yongchan Lee
- , Outi Haapanen
- & Volker Zickermann
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Article
| Open AccessManganese-driven CoQ deficiency
Across phylae, excess manganese disrupts energy metabolism by unclear mechanisms. Here, Diessl et al. report that failure of mitochondrial bioenergetics upon manganese overload is due to mismetallation of a diiron enzyme crucial for CoQ biosynthesis
- Jutta Diessl
- , Jens Berndtsson
- & Sabrina Büttner