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| Open AccessStructural insight on assembly-line catalysis in terpene biosynthesis
Substrate channeling can improve biosynthetic efficiency and has been implicated in the reactions of fusicoccadiene synthase. Here, the authors analyze this bifunctional enzyme complex by cryoEM, cross-linking MS and integrative modeling, providing structural insights into how substrate channeling is achieved.
- Jacque L. Faylo
- , Trevor van Eeuwen
- & David W. Christianson
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Article
| Open AccessSugar phosphate activation of the stress sensor eIF2B
The activity of translation initiation factor eIF2B is known to be modulated through stress-responsive phosphorylation of its substrate eIF2. Here, the authors uncover the regulation of eIF2B by the binding of sugar phosphates, suggesting a link between nutrient status and the rate of protein synthesis.
- Qi Hao
- , Jin-Mi Heo
- & Carmela Sidrauski
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Article
| Open AccessClostridioides difficile specific DNA adenine methyltransferase CamA squeezes and flips adenine out of DNA helix
Clostridioides difficile adenine methyltransferase A (CamA) is required for the sporulation and colonization of the pathogen that causes gastrointestinal infections. Here, the authors characterise CamA kinetically and present its crystal structure bound to the DNA recognition sequence, which reveals DNA distortions including bending and the flipping of the target adenine out of the DNA helix, as well as protein conformational changes upon cofactor binding.
- Jujun Zhou
- , John R. Horton
- & Xiaodong Cheng
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Article
| Open AccessGenome information processing by the INO80 chromatin remodeler positions nucleosomes
DNA sequence preferences or statistical positioning of histones has not explained genomic patterns of nucleosome organisation in vivo. Here, the authors establish DNA shape/mechanics as key elements that have evolved together with binding sites of DNA sequence-specific barriers so that such information directs nucleosome positioning by chromatin remodelers.
- Elisa Oberbeckmann
- , Nils Krietenstein
- & Sebastian Eustermann
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Article
| Open AccessExtreme parsimony in ATP consumption by 20S complexes in the global disassembly of single SNARE complexes
Fueled by ATP hydrolysis in N-ethylmaleimide sensitive factor (NSF), the 20S complex disassembles SNARE complexes in a single unravelling step. Here authors use single-molecule methods to show cooperativity between the NSF and SNARE complex, which prevents ATP consumption without productive disassembly.
- Changwon Kim
- , Min Ju Shon
- & Tae-Young Yoon
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Article
| Open AccessRuler elements in chromatin remodelers set nucleosome array spacing and phasing
Although chromatin remodelers have been shown to align nucleosome arrays to barriers and to generate spacing regularity among nucleosomes within arrays, it has remained unclear how the distance to barrier and the spacing length are determined in absolute terms. Here, the authors reveal that remodelers contain a ‘ruler’ element that sets remodeler-specific alignment and spacing distances when generating nucleosome arrays.
- Elisa Oberbeckmann
- , Vanessa Niebauer
- & Philipp Korber
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Article
| Open AccessCaerulomycin and collismycin antibiotics share a trans-acting flavoprotein-dependent assembly line for 2,2’-bipyridine formation
Caerulomycins and collismycins are two types of 2,2’-bipyridine natural products that are biosynthesized via a hybrid NRPS-PKS pathway, but the details of their biosynthesis were unknown. Here, the authors elucidate their biosynthetic pathways, validate the generality of 2,2’-bipyridine formation, and clarify the process for 2,2’-bipyridine furcation.
- Bo Pang
- , Rijing Liao
- & Wen Liu
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Article
| Open AccessStructural basis for allosteric regulation of Human Topoisomerase IIα
Type 2 DNA topoisomerases (Top2) regulates DNA topology during DNA replication, transcription, and chromosome segregation. Here the authors describe a complete structure of the catalytic core of the human Topo IIα bound to DNA and etoposide, providing insight into the regulation of Topo IIα activities and how opening of the DNA-gate is spatially connected to the ATPase domain.
- Arnaud Vanden Broeck
- , Christophe Lotz
- & Valérie Lamour
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Article
| Open AccessMechanism for DPY30 and ASH2L intrinsically disordered regions to modulate the MLL/SET1 activity on chromatin
Regulation of the MLL family of histone H3K4 methyltransferases on the nucleosome core particle (NCP) remains largely unknown. Here the authors show that intrinsically disordered regions of ASH2L and DPY30 restrict the rotational dynamics of MLL1 on the NCP, allowing more efficient enzyme-substrate engagement and higher H3K4 trimethylation activity.
- Young-Tae Lee
- , Alex Ayoub
- & Yali Dou
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Article
| Open AccessStructure-based engineering of substrate specificity for pinoresinol-lariciresinol reductases
Pinoresinol–lariciresinol reductases (PLRs) are enzymes involved in the lignan biosynthesis. Here, crystal structures of three PLRs in the apo, substrate-bound and product-bound states, and accompanying mutagenesis provide insight into PLRs catalytic mechanism and suggest a strategy for PLR engineering.
- Ying Xiao
- , Kai Shao
- & Wansheng Chen
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Article
| Open AccessMolecular basis for the allosteric activation mechanism of the heterodimeric imidazole glycerol phosphate synthase complex
The allosteric regulation of the bienzyme complex imidazole glycerol phosphate synthase (HisFH) remains to be elucidated. Here, the authors provide structural insights into the dynamic allosteric mechanism by which ligand binding to the cyclase and glutaminase active sites of HisFH regulate enzyme activation.
- Jan Philip Wurm
- , Sihyun Sung
- & Remco Sprangers
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Article
| Open AccessFollowing replicative DNA synthesis by time-resolved X-ray crystallography
DNA polymerases are the key enzymes responsible for DNA replication and repair. Here the authors reveal through time-lapsed images of X-ray crystal structures that translocation precedes phosphodiester bond formation in the mechanism of DNA synthesis.
- Nicholas Chim
- , Roman A. Meza
- & John C. Chaput
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Article
| Open AccessStructural visualization of transcription activated by a multidrug-sensing MerR family regulator
The MerR family of transcriptional regulators, such as EcmrR, activate promoters with a structure that is suboptimal for recognition by RNA polymerase holoenzyme. Structural insights into the EcmrR-dependent transcription process elucidate the mechanisms enabling optimal promoter recognition and transition from initiation to elongation.
- Yang Yang
- , Chang Liu
- & Bin Liu
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Article
| Open AccessStructural coordination between active sites of a CRISPR reverse transcriptase-integrase complex
In some systems, a single protein comprising reverse transcriptase (RT), integrase and maturase enables concerted sequence integration and crRNA production. Here, analyses including the structure of a Cas6-RT-Cas1—Cas2 complex suggest coordination between all three active sites and capacity to acquire CRISPR sequences from RNA and DNA substrates.
- Joy Y. Wang
- , Christopher M. Hoel
- & Jennifer A. Doudna
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Article
| Open AccessStructures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity
Non-ribosomal peptide synthetases (NRPSs) are multi-modular enzymes assembling complex natural products. Here, the structures of a Thermobifida fusca NRPS condensation domain bound to the substrate-bearing peptidyl carrier protein (PCP) domain provide insight into the mechanisms of substrate selectivity and engagement within the catalytic pocket.
- Thierry Izoré
- , Y. T. Candace Ho
- & Max J. Cryle
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Article
| Open AccessSpecificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD
The ER chaperone BiP is critical for the unfolded protein response and tightly regulated through reversible AMPylation by FICD, but the structural basis is unknown. Here the authors use thiol-reactive nucleotide derivatives to stabilize the transient FICD:BiP complex and determine its crystal structure.
- Joel Fauser
- , Burak Gulen
- & Aymelt Itzen
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Article
| Open AccessCrystal structures of an E1–E2–ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification
The molecular mechanism of ubiquitin transfer from E1 to E2 enzymes is still unclear. By solving the crystal structure of a covalently trapped E1–E2–ubiquitin thioester mimetic, the authors identify two conformations of this complex which suggest an affinity switch mechanism for thioester transfer.
- Lingmin Yuan
- , Zongyang Lv
- & Shaun K. Olsen
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Article
| Open AccessBiased cytochrome P450-mediated metabolism via small-molecule ligands binding P450 oxidoreductase
P450 oxidoreductase (POR) selectively activates numerous cytochromes P450 (CYP), crucial for metabolism of drugs, steroids and xenobiotics and natural product biosynthesis. Here, the authors identify ligands that bind POR and bias its specificity towards CYP redox partners, activating distinct metabolic cascades in cells.
- Simon Bo Jensen
- , Sara Thodberg
- & Nikos S. Hatzakis
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Article
| Open AccessActivation mechanism of a small prototypic Rec-GGDEF diguanylate cyclase
As part of two-component systems, diguanylate cyclases (DGCs) are activated by phosphorylation. Structural and computational analyses of DgcR, a model DGC, reveal the phosphorylation-induced conformational changes and the activation mechanism likely shared by many DGCs with N-terminal coiled-coil linkers.
- Raphael D. Teixeira
- , Fabian Holzschuh
- & Tilman Schirmer
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Article
| Open AccessProtein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1
The glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting enzyme in the hexosamine pathway producing uridine 5’-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), an essential glycosylation precursor. Here, the authors dissect the mechanisms of GFAT-1 regulation by protein kinase A (PKA)-mediated phosphorylation.
- Sabine Ruegenberg
- , Felix A. M. C. Mayr
- & Martin S. Denzel
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Article
| Open AccessBiochemical and structural characterization of the BioZ enzyme engaged in bacterial biotin synthesis pathway
Biotin is an essential enzyme cofactor and two pathways for the generation of the biotin precursor pimeloyl-ACP are known. Here, the authors identify and characterize a third pathway for biotin precursor synthesis involving BioZ and they also present the Agrobacterium tumefaciens BioZ crystal structure.
- Sitao Zhang
- , Yongchang Xu
- & Youjun Feng
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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 AccessWatching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide
How DNA polymerases discriminate against oxidized and undamaged nucleotides during DNA repair is not fully understood. Here, the authors reveal high-resolution timelapse X-ray crystallography snapshots of DSB repair polymerase μ undergoing DNA synthesis providing mechanistic insights into the process.
- Joonas A. Jamsen
- , Akira Sassa
- & Samuel H. Wilson
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Article
| Open AccessCrystal structures of human MGST2 reveal synchronized conformational changes regulating catalysis
Microsomal glutathione S-transferase 2 (MGST2) produces leukotriene C4, an intracrine mediator of cell death. Structural, biochemical and computational analyses of human MGST2 suggest a mechanism employed by the enzyme to restrict catalysis to only one active site within the MGST2 trimer.
- Madhuranayaki Thulasingam
- , Laura Orellana
- & Jesper Z. Haeggström
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Article
| Open AccessStructural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes
The phosphatidylinositol-3-phosphate (PI3P) is generated by the lipid kinase VPS34, in the context of VPS34 complex I on autophagosomes or complex II on endosomes. Biochemical and structural analyses provide insights into the mechanism of both VPS34 complexes recruitment to and activation on membranes by specific Rab GTPases.
- Shirley Tremel
- , Yohei Ohashi
- & Roger L. Williams
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Article
| Open AccessThe methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain
The methylation of Histone 3 at Lysine 36 (H3K36) has been implicated in the regulation of transcription and coupled processes such as mRNA splicing. Here the authors show that the histone methyltransferase SETD2 interacts with hnRNP L to mediate the crosstalk between the transcription and splicing machineries.
- Saikat Bhattacharya
- , Michaella J. Levy
- & Jerry L. Workman
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Article
| Open AccessEnzymatic spiroketal formation via oxidative rearrangement of pentangular polyketides
Rubromycin family of natural products belongs to aromatic polyketides with diverse bioactivities, but details of their biosynthesis are limited. Here, the authors report the complete in vitro reconstitution of enzymatic formation of the spiroketal moiety of rubromycin polyketides, driven by flavin-dependent enzymes, and characterize reaction intermediates.
- Britta Frensch
- , Thorsten Lechtenberg
- & Robin Teufel
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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 AccessStructure-guided insights into heterocyclic ring-cleavage catalysis of the non-heme Fe (II) dioxygenase NicX
2,5-Dihydroxypyridine dioxygenase NicX uses a mononuclear non-heme Fe(II) to catalyze the oxidative pyridine ring cleavage of the pollutant 2,5-hydroxypyridine. Here, the authors report crystal structures of NicX, identify residues involved in substrate stabilization and Fe(II) coordination, and propose the catalytic mechanism of NicX.
- Gongquan Liu
- , Yi-Lei Zhao
- & Ping Xu
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Article
| Open AccessDiscovery of exolytic heparinases and their catalytic mechanism and potential application
Exolytic heparinases are needed for sequencing of heparin and heparan sulfate (HP), but have not yet been reported. Here, the authors identify exolytic heparinases from different bacteria and show that the heparinases preferentially digest HP, determine the crystal structure of the exoheparinase BlexoHep and perform sequencing of HP octasaccharides using the enzyme.
- Qingdong Zhang
- , Hai-Yan Cao
- & Fuchuan Li
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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 AccessRING domains act as both substrate and enzyme in a catalytic arrangement to drive self-anchored ubiquitination
The mechanism by which RING E3-anchored ubiquitin chains are formed is not well understood. Here, the authors solve a crystal structure of the RING E3 enzyme TRIM21 trapped in the process of self-anchored chain elongation and provide biochemical and cellular insights into the mechanism of ubiquitin conjugation.
- Leo Kiss
- , Dean Clift
- & Leo C. James
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Article
| Open AccessFast acting allosteric phosphofructokinase inhibitors block trypanosome glycolysis and cure acute African trypanosomiasis in mice
Glycolytic enzymes are challenging drug targets due to their highly conserved active sites and phosphorylated substrates. Here, the authors identify fast acting allosteric inhibitors of Trypanosoma brucei phosphofructokinase that block trypanosome glycolysis and provide cure evidence in murine model.
- Iain W. McNae
- , James Kinkead
- & Malcolm D. Walkinshaw
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Article
| Open AccessMechanisms of feedback inhibition and sequential firing of active sites in plant aspartate transcarbamoylase
Aspartate transcarbamoylase acts in de novo pyrimidine biosynthesis and in plants is regulated by feedback inhibition via uridine 5-monophosphate (UMP). Here Bellin et al. describe the structural basis for this feedback inhibition, showing that UMP blocks the active site by binding to a plant specific UMP recognition loop.
- Leo Bellin
- , Francisco Del Caño-Ochoa
- & Santiago Ramón-Maiques
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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 AccessThe mechanism of the nucleo-sugar selection by multi-subunit RNA polymerases
RNA and DNA polymerases need to discriminate efficiently against closely related nucleotide triphosphate substrates. Here, the authors show that a conserved Arg residue is the major determinant of selectivity against deoxyribonucleoside substrates by multisubunit RNA polymerases.
- Janne J. Mäkinen
- , Yeonoh Shin
- & Georgiy A. Belogurov
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Article
| Open AccessDiscovery of an ene-reductase for initiating flavone and flavonol catabolism in gut bacteria
Flavonoids are abundant polyphenols in plants but it is not well understood how their metabolism is initiated by microbes in the human gut. Here, the authors identify and characterise an ene-reductase from the gut bacterium, Flavonifractor plautii ATCC 49531 that catalyses the hydrogenation of the C2–C3 double bond of flavones and flavonols and present its crystal structure.
- Gaohua Yang
- , Sen Hong
- & Yang Gu
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Article
| Open AccessHistone Parylation factor 1 contributes to the inhibition of PARP1 by cancer drugs
PARP1 is the target of clinically approved anti-cancer drugs whose in vivo efficacy has been hard to predict. Here the authors show how an altered active site formed between PARP1 and Histone PARylation Factor 1 (HPF1) changes the efficacy of some of these inhibitors.
- Johannes Rudolph
- , Genevieve Roberts
- & Karolin Luger
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Article
| Open AccessA safety cap protects hydrogenase from oxygen attack
[FeFe]-hydrogenases catalyze the conversion of protons and electrons to molecular hydrogen, but upon exposure to oxygen, their catalytic cofactor is irreversibly inactivated. Here, the authors determine the crystal structure of hydrogenase CbA5H and identify a cysteine residue, which acts as a safety cap that shields the active site from oxygen.
- Martin Winkler
- , Jifu Duan
- & Thomas Happe
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Article
| Open AccessAPE1 distinguishes DNA substrates in exonucleolytic cleavage by induced space-filling
The exonuclease Apurinic/apyrimidinic endonuclease 1 (APE1) is important for processing matched/mismatched terminus during DNA repair. Here the authors resolve the X-ray crystallography structures of APE1 with varied dsDNAs substrates to assay the nuclease activity.
- Tung-Chang Liu
- , Chun-Ting Lin
- & Yu-Yuan Hsiao
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Article
| Open AccessRab1-AMPylation by Legionella DrrA is allosterically activated by Rab1
The Legionella effector DrrA AMPylates the host protein Rab1 during infection, but the mechanism is still under debate. Here, the authors provide structural insights into the low-affinity DrrA:Rab1 interaction, showing that Rab1 allosterically activates DrrA through a non-conventional binding mechanism.
- Jiqing Du
- , Marie-Kristin von Wrisberg
- & Aymelt Itzen
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Article
| Open AccessCrystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism
Steroid 5α-reductase 2 (SRD5A2), a testosterone metabolism enzyme, is implicated in human disease. Structural and biochemical analyses of PbSRD5A, a bacterial homolog, reveal SRD5A2 substrate binding pocket and provide framework for the design of new drugs targeting this enzyme.
- Yufei Han
- , Qian Zhuang
- & Ruobing Ren
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Article
| Open AccessTwo distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations
Family 43 glycoside hydrolases (GH43) are involved in the breakdown of hemicellulose. Functional, structural and computational characterization of a GH43 enzyme, including a snapshot of an active Michaelis complex, reveal the hydrolysis mechanism and suggest two possible reaction pathways.
- Mariana A. B. Morais
- , Joan Coines
- & Mario T. Murakami
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Article
| Open AccessStructure of a full-length bacterial polysaccharide co-polymerase
Lipopolysaccharides, important components of the bacterial cell envelope, are synthesized at the inner membrane by the Wzx/Wzy-dependent assembly pathway. A cryo-EM structure of an intact E. coli WzzB, the polysaccharide co-polymerase component of this pathway, reveals details of the transmembrane, cytoplasmic domains and a conserved a proline-rich segment proximal to the C-terminal transmembrane helix.
- Benjamin Wiseman
- , Ram Gopal Nitharwal
- & Martin Högbom
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Article
| Open AccessInsights into genome recoding from the mechanism of a classic +1-frameshifting tRNA
Genome recoding with quadruplet codons requires a +1-frameshift-suppressor tRNA able to insert an amino acid at quadruplet codons of interest. Here the authors identify the mechanisms resulting in +1 frameshifting and the steps of the elongation cycle in which it occurs.
- Howard Gamper
- , Haixing Li
- & Ya-Ming Hou
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Article
| Open AccessEngineering and elucidation of the lipoinitiation process in nonribosomal peptide biosynthesis
Nonribosomal lipopeptides contain an acyl chain important for bioactivity, but its incorporation into the peptidyl backbone, mediated by the starter condensation (Cs) domain of nonribosomal peptide synthases, is not fully understood. Here, the authors show that acyl chains of different lengths can be obtained by engineering Cs domains and identify residues that determine the selectivity for acyl chains.
- Lin Zhong
- , Xiaotong Diao
- & Xiaoying Bian
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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 AccessCatalytically potent and selective clusterzymes for modulation of neuroinflammation through single-atom substitutions
Artificial enzymes with reprogrammed and augmented catalytic activity and substrate selectivity have emerged to tackle limitations of noble metals or transition metal oxides. Here, the authors report Au25 clusterzymes which are endowed with high catalytic activity and selectivity in a range of enzyme-mimicking reactions.
- Haile Liu
- , Yonghui Li
- & Xiao-Dong Zhang
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Article
| Open AccessThe δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling
The bacterial helicase-like transcription factor HelD interacts with the RNA polymerase (RNAP) and together with the RNAP δ subunit enhances RNAP cycling. Here, the authors present the cryo-EM structures of the monomeric and dimeric Bacillus subtilis RNAP-δ-HelD complexes and suggest a model for HelD/δ-mediated RNAP recycling and putative hibernation.
- Hao-Hong Pei
- , Tarek Hilal
- & Markus C. Wahl