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| Open AccessHeme biosynthesis depends on previously unrecognized acquisition of iron-sulfur cofactors in human amino-levulinic acid dehydratase
Heme biosynthesis depends on iron-sulfur (Fe-S) cluster biogenesis but the molecular connection between these pathways is not fully understood. Here, the authors show that the heme biosynthesis enzyme ALAD contains an Fe-S cluster, disruption of which reduces ALAD activity and heme production in human cells.
- Gang Liu
- , Debangsu Sil
- & Tracey Ann Rouault
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Article
| Open AccessReverse C-glycosidase reaction provides C-nucleotide building blocks of xenobiotic nucleic acids
C-nucleosides are analogues of the canonical N-nucleosides and, despite their synthetic value, biocatalysis has not targeted them yet. Here, the authors report a pseudouridine monophosphate C-glycosidase enzyme for selective 5-β-C-glycosylation of uracil and its derivatives from pentose 5- phosphate substrates.
- Martin Pfeiffer
- & Bernd Nidetzky
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Article
| Open AccessMolecular basis of regio- and stereo-specificity in biosynthesis of bacterial heterodimeric diketopiperazines
Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are bioactive natural products that are difficult to access chemically. Here, the authors identify a family of three related HTDKP-forming cytochrome P450s and engineer key amino acid residues to produce distinct diketopiperazines frameworks.
- Chenghai Sun
- , Zhenyao Luo
- & Xudong Qu
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Article
| Open AccessStructure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation
AlfC transfucosidase is used to modulate fucosylation of glycans decorating monoclonal antibodies. Herein, structural and biophysical characterization reveals the enzymatic mechanism of AlfC and a blueprint for the design of AlfC mutants with novel specificities and functions.
- Erik H. Klontz
- , Chao Li
- & Eric J. Sundberg
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Article
| Open AccessDirect binding of TFEα opens DNA binding cleft of RNA polymerase
How clamp conformation is regulated in the transcription cycle of stalk-containing archaeal and eukaryotic RNA polymerase (RNAP) systems is still not well understood. Here, the authors combine cryo-EM, X-ray crystallography and photo-crosslinking assays to structurally characterise RNAP, the RNAP-TFEα binary and RNAP-TFEα-promoter DNA ternary complexes from the archaea Thermococcus kodakarensis and enables them to describe the dynamic conformational changes of the general transcription factor TFEα and RNAP during the early stage of transcription cycle.
- Sung-Hoon Jun
- , Jaekyung Hyun
- & Katsuhiko S. Murakami
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Article
| Open AccessMechanism and inhibition of Streptococcus pneumoniae IgA1 protease
Pathogenic IgA1 metalloproteases block the initial host immune response by cleaving host IgA1. Using cryoEM, the authors here provide structural insights into the substrate recognition mechanism of Streptococcus pneumoniae IgA1 protease, and develop a protease-inhibiting antibody.
- Zhiming Wang
- , Jeremy Rahkola
- & Elan Eisenmesser
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Article
| Open AccessStructural basis for assembly of non-canonical small subunits into type I-C Cascade
Type I-C Cascade (the CRISPR-associated complex for antiviral defense) is a minimal system, comprising only three unique Cas proteins. Cryo-EM structure of the Desulfovibrio vulgaris type I-C Cascade reveals the molecular mechanisms that underlie RNA-directed complex assembly.
- Roisin E. O’Brien
- , Inês C. Santos
- & David W. Taylor
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Article
| Open AccessDinucleosome specificity and allosteric switch of the ISW1a ATP-dependent chromatin remodeler in transcription regulation
Here the authors show that the preference of yeast chromatin remodeler ISW1a for dinucleosomes hinges on conformational changes that occur in the transition from binding mononucleosomes to dinucleosomes. These changes are critical for ISW1a organizing chromatin at promoters and regulating transcription in conjunction with other chromatin remodelers.
- Saurabh K. Bhardwaj
- , Solomon G. Hailu
- & Blaine Bartholomew
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Article
| Open AccessCrystallographic structure of wild-type SARS-CoV-2 main protease acyl-enzyme intermediate with physiological C-terminal autoprocessing site
The SARS-CoV-2 main protease (Mpro) is one of two cysteine proteases essential for viral replication. Here, the authors determine the crystal structure of an Mpro acyl intermediate with its native C-terminal autocleavage sequence and the structure of a product bound active site mutant (C145A), which are of interest for antiviral drug development.
- Jaeyong Lee
- , Liam J. Worrall
- & Natalie C. J. Strynadka
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Article
| Open Accessα-proteobacteria synthesize biotin precursor pimeloyl-ACP using BioZ 3-ketoacyl-ACP synthase and lysine catabolism
Biosynthesis of biotin precursor, pimelate moiety, is elucidated in Escherichia coli and Bacillus subtilis, but not understood in alphaproteobacteria. Here, the authors show that BioZ, a 3-ketoacyl-ACP synthase, catalayses pimeloyl-thioester synthesis in alphaproteobacteria using malonyl-ACP and glutaryl-CoA that is derived from lysine degradation.
- Yuanyuan Hu
- & John E. Cronan
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Article
| Open AccessPhase separation by the polyhomeotic sterile alpha motif compartmentalizes Polycomb Group proteins and enhances their activity
The conserved SAM motif of Polycomb Repressive Complex 1 subunit Ph has been shown to play an important role in chromatin organization. Here, the authors study the effect of Ph SAM on chromatin in vitro, showing that it induces the formation of concentrated, phase-separated condensates, which enhance the ubiquitin ligase activity of PRC1.
- Elias Seif
- , Jin Joo Kang
- & Nicole J. Francis
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Article
| Open AccessAllomorphy as a mechanism of post-translational control of enzyme activity
β-phosphoglucomutase (βPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for catabolism of trehalose and maltose. Coupled analyses of multiple βPGM structures and enzymatic activity lead to the proposal of allomorphy — a post-translational mechanism controlling enzyme activity.
- Henry P. Wood
- , F. Aaron Cruz-Navarrete
- & Jonathan P. Waltho
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Article
| Open AccessDivergent architecture of the heterotrimeric NatC complex explains N-terminal acetylation of cognate substrates
The conserved eukaryotic heterotrimeric NatC complex co-translationally acetylates the N-termini of numerous target proteins. Here, the authors provide insights into the catalytic mechanism of NatC by determining the crystal structures of Saccharomyces cerevisiae NatC in the absence and presence of cofactors and peptide substrates and reveal the molecular basis of substrate binding by further biochemical analyses.
- Stephan Grunwald
- , Linus V. M. Hopf
- & Oliver Daumke
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Article
| Open AccessMolecular mechanism of the MORC4 ATPase activation
Human Microrchidia 4 (MORC4) ATPase has been implicated in acute and chronic pancreatitis, inflammatory disorders and cancer. Here the authors describe the structure–function relationship of MORC4 and define the molecular mechanism for MORC4 activation.
- Adam H. Tencer
- , Khan L. Cox
- & Tatiana G. Kutateladze
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Article
| Open AccessStructure of human steroid 5α-reductase 2 with the anti-androgen drug finasteride
Human steroid 5α-reductase 2 (SRD5A2) is an integral membrane enzyme and catalyzes 5α-reduction of testosterone to dihydrotestosterone. Structural analysis accompanied by computational and mutagenesis studies reveal the mechanisms of catalysis and inhibition by clinically relevant drugs targeting SRD5A2.
- Qingpin Xiao
- , Lei Wang
- & Cheng Zhang
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Article
| Open AccessMechanisms of telomerase inhibition by oxidized and therapeutic dNTPs
Telomerase enzymes add telomeric repeats to the end of linear chromosomes. Here the authors reveal mechanisms by which oxidized dNTPs and therapeutic dNTPs inhibit telomerase-mediated telomere elongation.
- Samantha L. Sanford
- , Griffin A. Welfer
- & Patricia L. Opresko
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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 AccessOxepinamide F biosynthesis involves enzymatic d-aminoacyl epimerization, 3H-oxepin formation, and hydroxylation induced double bond migration
Oxepinamides are a class of fungal oxepins with biological activities. Here, the authors elucidate the biosynthetic pathway of oxepinamide F from Aspergillus ustus and show that it involves enyme-catalysed oxepin ring formation, hydroxylation-induced double bond migration, epimerization and methylation.
- Liujuan Zheng
- , Haowen Wang
- & Shu-Ming Li
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Article
| Open AccessStructural and mechanistic basis of capsule O-acetylation in Neisseria meningitidis serogroup A
Neisseria meningitidis capsular polysaccharide (CPS) is a major virulence factor and vaccine formulations against Neisseria meningitidis serogroup A (NmA) contain O-acetylated CPS. Here, the authors provide mechanistic insights into CPS O-acetylation in NmA by determining the crystal structure of the O-acetyltransferase CsaC and NMR measurements further reveal that the CsaC-mediated reaction is regioselective for O3 and that the O4 modification results from spontaneous O-acetyl migration.
- Timm Fiebig
- , Johannes T. Cramer
- & Martina Mühlenhoff
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Article
| Open AccessElucidating the role of metal ions in carbonic anhydrase catalysis
Metalloenzymes often show different catalytic activities in the presence of non-native metal ions. Here, the authors report structures of carbonic anhydrase bound to zinc and several other metal ions and demonstrate that metal ion coordination geometries directly impact catalytic activity of the enzyme.
- Jin Kyun Kim
- , Cheol Lee
- & Chae Un Kim
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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 AccessStructural mechanism for replication origin binding and remodeling by a metazoan origin recognition complex and its co-loader Cdc6
The origin recognition complex (ORC) is essential for loading the Mcm2–7 replicative helicase onto DNA during DNA replication initiation. Here, the authors describe several cryo-electron microscopy structures of Drosophila ORC bound to DNA and its cofactor Cdc6 and also report an in vitro reconstitution system for Drosophila Mcm2–7 loading, revealing unexpected features of ORC’s DNA binding and remodeling mechanism during Mcm2–7 loading.
- Jan Marten Schmidt
- & Franziska Bleichert
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Article
| Open AccessMolecular basis for DNA repair synthesis on short gaps by mycobacterial Primase-Polymerase C
Mycobacteria Prim-PolC performs short gap synthesis following removal of lesions during excision repair. Here the authors resolve crystal structures of pre- and post-catalytic Prim-PolC complexes bound to gapped DNA substrates to define its mechanism.
- Nigel C. Brissett
- , Katerina Zabrady
- & Aidan J. Doherty
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Article
| Open AccessOTUD5 cooperates with TRIM25 in transcriptional regulation and tumor progression via deubiquitination activity
The mechanisms by which deubiquitinases modulate tumour progression are not fully understood. Here, the authors perform an RNAi screen and identify that the deubiquitinase OTUD5 suppresses cancer growth in a TRIM25 dependent manner, which in turn controls the expression of tumour suppressor protein, PML.
- Fangzhou Li
- , Qianqian Sun
- & Wenhui Zhao
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Article
| Open AccessKey role of quinone in the mechanism of respiratory complex I
Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the respiratory chain in bacteria and mitochondria. Here, the authors present cryo-EM and crystal structures of T. thermophilus complex I in different conformational states and further analyse them by Normal Mode Analysis and molecular dynamics simulations and conclude that quinone redox reactions are important for the coupling mechanism of complex I.
- Javier Gutiérrez-Fernández
- , Karol Kaszuba
- & Leonid A. Sazanov
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Article
| Open AccessVisualizing the enzyme mechanism of mevalonate diphosphate decarboxylase
Mevalonate diphosphate decarboxylase (MDD) is a key enzyme in the mevalonate pathway and catalyses the decarboxylation of mevalonate-5-diphosphate to isopentenyl diphosphate. Here, the authors provide insights into the conformational changes that occur during substrate binding of MDD and the subsequent enzymatic reaction steps by determining the substrate and intermediate bound crystal structures of Enterococcus faecalis MDD.
- Chun-Liang Chen
- , Lake N. Paul
- & Cynthia V. Stauffacher
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Article
| Open AccessDiscovery of the cryptic function of terpene cyclases as aromatic prenyltransferases
Terpene cyclases catalyze the formation of diverse hydrocarbon scaffolds found in terpenoids. Here, the authors report the cryptic function of class I terpene cyclases as aromatic prenyltransferases and the universality of this cryptic feature is confirmed using enzymes from different sources.
- Haibing He
- , Guangkai Bian
- & Tiangang Liu
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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 AccessA distal regulatory region of a class I human histone deacetylase
Human Histone Deacetylases (HDACs) regulate gene expression and are important drug targets. Here, the authors combine NMR measurements, enzymatic assays and molecular dynamics simulations and show that HDAC8 samples a catalytically active and an inactive state and further demonstrate that mutations and ligand binding alter the populations of the two states, which is of interest for inhibitor design.
- Nicolas D. Werbeck
- , Vaibhav Kumar Shukla
- & D. Flemming Hansen
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Article
| Open AccessFunctional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer
RNF4 is a prototypical single-subunit E3 enzyme that can bind both substrate and ubiquitin-loaded E2. Here, the authors show that the RNF4 N-terminal region, although lacking a defined secondary structure, maintains a compact global conformation to facilitate ubiquitin transfer to the substrate.
- Paul Murphy
- , Yingqi Xu
- & Ronald T. Hay
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Article
| Open AccessDuplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase
During eukaryotic chromosome replication cells utilize ring-shaped CMG helicase that separates the two strands of the DNA double helix. Here the authors reveal that CMG helicase activity is inhibited by duplex DNA engagement at the fork, which is relieved by binding of RPA to the lagging-strand template.
- Hazal B. Kose
- , Sherry Xie
- & Hasan Yardimci
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Article
| Open AccessDNA sequence-dependent activity and base flipping mechanisms of DNMT1 regulate genome-wide DNA methylation
DNA methylation is one of the major epigenetic mechanisms that critically influence gene expression, genomic stability and cell differentiation. Here, the authors study DNMT1 in complex with DNA substrates and systematically analyze the mechanism and specificity of DNMT1.
- Sabrina Adam
- , Hiwot Anteneh
- & Albert Jeltsch
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Article
| Open AccessEngineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA
Engineered high-fidelity Cas9s have increased discrimination against off-targets. Kinetic analyses of Cas9-HF1 and HypaCas9 engineered Cas9 variants show that their DNA cleavage is impaired by more than 100- fold, which leads to release rather than cleavage of a bound off-target substrate.
- Mu-Sen Liu
- , Shanzhong Gong
- & David W. Taylor
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Article
| Open AccessMolecular basis for the distinct functions of redox-active and FeS-transfering glutaredoxins
Glutaredoxins are a family of essential enzymes divided into two major classes with either a CGFS or a CxxC active site, of which only the latter exhibits oxidoreductase activity. Here the authors address the structural basis for the functional difference between the two classes of glutaredoxins.
- Daniel Trnka
- , Anna D. Engelke
- & Christopher Horst Lillig
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Article
| Open AccessR-loop induced G-quadruplex in non-template promotes transcription by successive R-loop formation
G-quadruplex (G4) forming sequences are highly enriched in the human genome and function as important regulators of diverse range of biological processes. Here the authors show that while G4 structures on template strand block transcription, folding on the non-template strand enhances transcription by means of successive R-loop formation.
- Chun-Ying Lee
- , Christina McNerney
- & Sua Myong
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Article
| Open AccessComprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms
In mammals, DNA methylation patterns are established by two de novo DNA methyltransferases, DNMT3A and DNMT3B. Here the authors report the crystal structures of DNMT3B in complex with both CpG and CpA DNA, providing insight into the substrate-recognition mechanism underpinning the divergent genomic methylation activities of DNMT3A and DNMT3B.
- Linfeng Gao
- , Max Emperle
- & Jikui Song
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Article
| Open AccessThe receptor PTPRU is a redox sensitive pseudophosphatase
Receptor-linked protein tyrosine phosphatases (RPTPs) usually contain an active membrane proximal domain and an inactive pseudophosphatase domain. Here, the authors characterize an RPTP with two pseudophosphatase domains, providing evidence that it may act as a decoy receptor for substrate sequestration.
- Iain M. Hay
- , Gareth W. Fearnley
- & Janet E. Deane
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Article
| Open AccessStructure and catalytic regulation of Plasmodium falciparum IMP specific nucleotidase
Plasmodium falciparum IMP-specific 5′-nucleotidase 1 (PfISN1) is of interest as a potential malaria drug target. Here, the authors report that IMP is a substrate, and ATP an allosteric activator, of PfISN1 and present PfISN1 crystal structures in the ligand-free state and bound to either IMP or ATP.
- Loïc Carrique
- , Lionel Ballut
- & Nushin Aghajari
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Article
| Open AccessCrystal structures of SAMHD1 inhibitor complexes reveal the mechanism of water-mediated dNTP hydrolysis
SAMHD1 catalyses the hydrolysis of dNTPs into 2′-deoxynucleosides and triphosphate and is an important regulator of cellular dNTP homeostasis. Here, the authors provide insights into the catalytic mechanism of SAMHD1 by performing kinetic measurements and determining crystal structures of α-β-imido-dNTP inhibitor complexes, which reveal a bi-metallic iron-magnesium centre and catalytic hydroxyl molecule in the active site of the enzyme.
- Elizabeth R. Morris
- , Sarah J. Caswell
- & Ian A. Taylor
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Article
| Open AccessStructure of the polymerase ε holoenzyme and atomic model of the leading strand replisome
DNA polymerase epsilon (Pol ε) is responsible for leading strand synthesis during DNA replication. Here the authors use Cryo-EM to describe the architecture of the Pol ε holoenzyme and to provide an atomic model for the leading strand replisome.
- Zuanning Yuan
- , Roxana Georgescu
- & Huilin Li
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Article
| Open AccessReal-time tracking reveals catalytic roles for the two DNA binding sites of Rad51
Rad51 drives DNA strand exchange, the central reaction in recombinational DNA repair. Two sites of Rad51 are responsible for DNA binding, but the function of these sites has proven elusive. Here, the authors employ real-time assays to reveal catalytic roles for the two DNA binding sites of Rad51.
- Kentaro Ito
- , Yasuto Murayama
- & Hiroshi Iwasaki
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Article
| Open AccessAn iron (II) dependent oxygenase performs the last missing step of plant lysine catabolism
Hydroxyglutarate synthase (HglS) converts 2-oxoadipate to D-2- hydroxyglutarate during lysine catabolism in bacteria. Here the authors use structural and biochemical approaches to show that HglS acts via successive decarboxylation and intramolecular hydroxylation and that homologous enzymes catalyze the final step of lysine catabolism in plants.
- Mitchell G. Thompson
- , Jacquelyn M. Blake-Hedges
- & Jay D. Keasling
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Article
| Open AccessUbiquitin transfer by a RING E3 ligase occurs from a closed E2~ubiquitin conformation
The mechanism of ubiquitin transfer from the ubiquitin-conjugated E2 enzyme (E2~Ub) to the substrate is still under debate. Here, the authors use FRET assays to show that RING E3 ligases transfer ubiquitin to the substrate from a closed E2~Ub conformation.
- Emma Branigan
- , J. Carlos Penedo
- & Ronald T. Hay
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Article
| Open AccessHuman aminolevulinate synthase structure reveals a eukaryotic-specific autoinhibitory loop regulating substrate binding and product release
Mutation of the C-terminal extension of 5′-aminolevulinate synthase 2 (ALAS2) is the molecular cause for X-linked protoporphyria, but the underlying mechanism is unclear. Based on crystal structures and MD simulations of ALAS2, the authors here show how the C-terminal extension regulates ALAS2 activity.
- Henry J. Bailey
- , Gustavo A. Bezerra
- & Wyatt W. Yue
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Article
| Open AccessInsights into catalysis and regulation of non-canonical ubiquitination and deubiquitination by bacterial deamidase effectors
The bacterial effector MavC can ubiquitinate the host E2 enzyme UBE2N to dampen the host immune response. Here, the authors provide mechanistic insight into this non-canonical ubiquitination machinery and reveal the structural basis for the functional differences between MavC and its close homolog MvcA.
- Yong Wang
- , Qi Zhan
- & Pu Gao
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Article
| Open AccessStructural insight into the electron transfer pathway of a self-sufficient P450 monooxygenase
Self-sufficient cytochrome P450 monooxygenases, which contain all redox partners in a single polypeptide chain, are of interest for biotechnological applications. Here, the authors present the crystal structure of full-length Thermobispora bispora CYP116B46 and discuss the potential electron transfer pathway.
- Lilan Zhang
- , Zhenzhen Xie
- & Chun-Chi Chen
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Article
| Open AccessStringent control of the RNA-dependent RNA polymerase translocation revealed by multiple intermediate structures
During translocation by viral RNA-dependent RNA polymerases (RdRP), the template-product RNA duplex moves in an asymmetric manner. Here the authors describe several crystal structures of RdRP translocations intermediates that, along with enzymological data, provide a comprehensive view of RdRP translocation.
- Meihua Wang
- , Rui Li
- & Peng Gong
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Article
| Open AccessReal-time monitoring of PARP1-dependent PARylation by ATR-FTIR spectroscopy
The mechanism of PARP1-dependent poly-ADP-ribosylation in response to DNA damage is still under debate. Here, the authors use ATR-FTIR spectroscopy to provide time-resolved insights into the molecular details of this process under near physiological conditions.
- Annika Krüger
- , Alexander Bürkle
- & Aswin Mangerich
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Article
| Open AccessCryo-EM structures reveal intricate Fe-S cluster arrangement and charging in Rhodobacter capsulatus formate dehydrogenase
Rhodobacter capsulatus NAD+ dependent formate dehydrogenase (RcFDH) is a molybdoenzyme that catalyses the reversible oxidation of formate to carbon dioxide, and is of interest for biotechnological applications. Here the authors present the cryo-EM structures of RcFDH as isolated from R. capsulatus and in the reduced state with bound NADH, and discuss the enzyme mechanism.
- Christin Radon
- , Gerd Mittelstädt
- & Petra Wendler