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| Open AccessCrosstalk between CST and RPA regulates RAD51 activity during replication stress
During replication stress, the RPA protein complex coats single-stranded DNA to preclude RAD51 loading. Here, the authors show how RPA and CST crosstalk to regulate RAD51 activity.
- Kai-Hang Lei
- , Han-Lin Yang
- & Peter Chi
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Article
| Open AccessArginine methylation and ubiquitylation crosstalk controls DNA end-resection and homologous recombination repair
Post-translational modifications are critical for regulating the DNA damage response. Here, the authors identify a methylation-deubiquitination crosstalk between methyltransferase PRMT1 and deubiquitinase USP11, showing that the enzymes regulate each other’s functions in DNA repair.
- Maria Pilar Sanchez-Bailon
- , Soo-Youn Choi
- & Clare C. Davies
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Article
| Open AccessInhibition of Clostridium difficile TcdA and TcdB toxins with transition state analogues
The Clostridium difficile virulence factors TcdA and TcdB contain a glucosyltransferase domain (GTD), which has both glucohydrolase (GH) and glucosyltransferase (GT) activities. Here, the authors characterize the transition state features of the TcdA and TcdB GH reactions by measuring kinetic isotope effects and they identify two transition state analogues, isofagomine and noeuromycin that inhibit TcdA and TcdB. They also present the crystal structures of TcdB-GTD bound to these inhibitors and the reaction product UDP.
- Ashleigh S. Paparella
- , Briana L. Aboulache
- & Vern L. Schramm
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Article
| Open AccessSustained enzymatic activity and flow in crowded protein droplets
Living cells can harvest environmental energy to drive chemical processes. Here the authors design a minimal artificial system that achieves steady states at similar metabolic densities to microorganisms.
- Andrea Testa
- , Mirco Dindo
- & Paola Laurino
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Article
| Open AccessStructural basis for protein glutamylation by the Legionella pseudokinase SidJ
Legionella pneumophila (LP) employs the metaeffector SidJ to suppress the toxicity of SdeA and other LP SidE effector family members by catalysing the glutamylation of the catalytic Glu residue. Here, the authors present the cryo-EM structures of SidJ in complex with SdeA in two different states, which together with mutagenesis analysis provide insights into the substrate recognition and the mechanism of protein glutamylation by SidJ.
- Michael Adams
- , Rahul Sharma
- & Sagar Bhogaraju
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Article
| Open AccessPalmitoylation targets the calcineurin phosphatase to the phosphatidylinositol 4-kinase complex at the plasma membrane
Calcineurin — the Ca2+ regulated phosphatase and target of immunosuppressants — regulates GPCR-mediated phospholipid signaling at the plasma membrane. Here the authors show that CNAβ1 (a poorly studied isoform of the calcineurin catalytic subunit) is targeted to the plasma membrane through palmitoylation to dephosphorylate and promote PI4KA complex activity.
- Idil Ulengin-Talkish
- , Matthew A. H. Parson
- & Martha S. Cyert
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Article
| Open AccessThe δ subunit of F1Fo-ATP synthase is required for pathogenicity of Candida albicans
F1Fo-ATP synthase is a key enzyme for energy production in fungi. Here, the authors show that the δ subunit of the enzyme is required for Candida albicans lethal infection and represents a potential therapeutic target.
- Shuixiu Li
- , Yajing Zhao
- & Hong Zhang
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Article
| Open AccessHuman cell based directed evolution of adenine base editors with improved efficiency
Application of adenine base editors (ABE) has been precluded by low activity. Here the authors show the generation of a human cell based ABE directional screening system and identification of ABE variant (NG-ABEmax-KR) exhibiting a significant increase in activity for human and mouse genome manipulation.
- Junhao Fu
- , Qing Li
- & Feng Gu
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Article
| Open AccessThiocysteine lyases as polyketide synthase domains installing hydropersulfide into natural products and a hydropersulfide methyltransferase
Enzymes installing an intact hydropersulfide (-SSH) group into natural products have so far not been identified. Here, the authors report the characterization of an S-adenosyl methionine-dependent hydropersulfide methyltransferase (GnmP) for guangnanmycin biosynthesis, and identification of three SH domains within several NRPS-PKS assembly lines as thiocysteine lyases.
- Song Meng
- , Andrew D. Steele
- & Ben Shen
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Article
| Open AccessActivation mechanism of human soluble guanylate cyclase by stimulators and activators
Soluble guanylate cyclase (sGC) is a validated drug target for cardiovascular diseases. Here, the authors report structures of human sGC in complex with NO and sGC stimulators or activator, providing insight into the mechanism of sGC activation by pharmacological compounds.
- Rui Liu
- , Yunlu Kang
- & Lei Chen
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Article
| Open AccessUbiD domain dynamics underpins aromatic decarboxylation
Understanding the structure and dynamics of enzymes is important for a number of applications. Here, the authors report on the crystal structure of vanillic acid decarboxylase, and show how the dynamics of the UbiD superfamily enzymes relate to the covalent catalysis of aromatic (de)carboxylation.
- Stephen A. Marshall
- , Karl A. P. Payne
- & David Leys
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Article
| Open AccessToward scalable biocatalytic conversion of 5-hydroxymethylfurfural by galactose oxidase using coordinated reaction and enzyme engineering
5-Hydroxymethylfurfural (HMF) can be transformed to a range of industrially useful derivatives, such as 2,5-diformylfuran (DFF), but the reactions needed for efficient industrial production are hindered by several issues. Here, the authors perform reaction and enzyme engineering resulting in a galactose oxidase variant with high activity towards HMF, improved oxygen binding and high productivity.
- William R. Birmingham
- , Asbjørn Toftgaard Pedersen
- & Nicholas J. Turner
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Article
| Open AccessThe six steps of the complete F1-ATPase rotary catalytic cycle
F1Fo ATP synthase works using a rotary catalysis mechanism. Here, the authors report cryo-EM structures of Bacillus PS3 F1-ATPase encompassing the complete set of six states taken up during the catalytic cycle, including the binding- and catalytic-dwell states.
- Meghna Sobti
- , Hiroshi Ueno
- & Alastair G. Stewart
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Article
| Open AccessPALI1 facilitates DNA and nucleosome binding by PRC2 and triggers an allosteric activation of catalysis
The polycomb repressive complex 2 (PRC2) is a histone methyltransferase regulating cell differentiation and identity. Here, the authors show that the vertebrate-specific PRC2 accessory subunit PALI1 facilitates substrate binding by the complex and elucidate the allosteric mechanism of PALI1- mediated PRC2 activation.
- Qi Zhang
- , Samuel C. Agius
- & Chen Davidovich
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Article
| Open AccessMolecular insights into the endoperoxide formation by Fe(II)/α-KG-dependent oxygenase NvfI
Many endoperoxide-containing natural products have been isolated, but the biosynthesis of the endoperoxides remains unclear. Here, the authors report the structural and functional analysis of the NvfI endoperoxidase that catalyzes the formation of fumigatonoid A in the biosynthesis of novofumigatonin, and show that it does not employ tyrosyl radical in the reaction.
- Takahiro Mori
- , Rui Zhai
- & Ikuro Abe
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Article
| Open AccessComplete loss of H3K9 methylation dissolves mouse heterochromatin organization
Histone H3K9 methylation (H3K9me) states define repressed chromatin in eukaryotic cells. Here the authors reveal complete loss of all H3K9me in mammalian cells through successive deletion of H3K9 methyltransferase genes that results in the dissolution of heterochromatin and the derepression of nearly all repeat families.
- Thomas Montavon
- , Nicholas Shukeir
- & Thomas Jenuwein
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Article
| Open AccessStructural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins
Prenylated indole alkaloids contain spirooxindole rings with a 3R or 3S carbon stereocenter, which determines their bioactivities, but the biocatalytic mechanism controlling the 3R- or 3S-spirooxindole formation was unclear. Here, the authors report the biochemical and structural characterization of the oxygenase/semipinacolase CtdE that catalyses the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A.
- Zhiwen Liu
- , Fanglong Zhao
- & Xue Gao
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Article
| Open AccessLight-driven decarboxylative deuteration enabled by a divergently engineered photodecarboxylase
The chemical processes for the selective incorporation of deuterium into small molecules, of interest to organic and medicinal chemistry, are well established, while the enzymatic methods remain underdeveloped. Here, the authors use an enzymatic approach employing Chlorella variabilis NC64A photodecarboxylase that catalyses decarboxylative deuteration of various carboxylic acids with D2O, and identify enzyme variants that can employ substrates with different chain length acids.
- Jian Xu
- , Jiajie Fan
- & Qi Wu
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Article
| Open AccessPhysical constraints and functional plasticity of cellulases
Enzyme reactions at interfaces are common in both Nature and industrial applications but no general kinetic framework exists for interfacial enzymes. Here, the authors kinetically characterize 83 cellulases and identify a scaling relationship between ligand binding strength and maximal turnover, a so-called linear free energy relationship, which may help rationalize cellulolytic mechanisms and guide the selection of technical enzymes.
- Jeppe Kari
- , Gustavo A. Molina
- & Peter Westh
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Article
| Open AccessHakai is required for stabilization of core components of the m6A mRNA methylation machinery
The E3 ligase Hakai can interact with the m6A methylation machinery but its function is still unclear. Here, the authors show that Hakai is a conserved component of the m6A methyltransferase complex and provide functional and molecular insights into its role in regulating m6A levels in Drosophila.
- Praveen Bawankar
- , Tina Lence
- & Jean-Yves Roignant
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Article
| Open AccessMechanism of genome instability mediated by human DNA polymerase mu misincorporation
Pol μ performs gap-filling repair synthesis in the nonhomologous end joining (NHEJ) pathway. Here the authors provide crystal structures and kinetics of human Pol μ to reveal insights into the molecular mechanism of Pol μ during the process of dGTP misincorporation.
- Miao Guo
- , Yina Wang
- & Ye Zhao
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Article
| Open AccessCRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation
CAPPs are putative Primase-Polymerases associated with CRISPR-Cas operons. Here, the authors show CAPPs genetic and physical association with Cas1 and Cas2, their capacity to function as DNA-dependent DNA primases and DNA polymerases, and that Cas1-Cas2 complex adjacent to CAPP has bona fide spacer integration activity.
- Katerina Zabrady
- , Matej Zabrady
- & Aidan J. Doherty
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Article
| Open AccessEngineering the protein dynamics of an ancestral luciferase
Directed evolution commonly relies on point mutations but InDels frequently occur in evolution. Here the authors report a protein-engineering framework based on InDel mutagenesis and fragment transplantation resulting in greater catalysis and longer glow-type bioluminescence of the ancestral luciferase.
- Andrea Schenkmayerova
- , Gaspar P. Pinto
- & Jiri Damborsky
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Article
| Open AccessA metal ion orients SARS-CoV-2 mRNA to ensure accurate 2′-O methylation of its first nucleotide
The SARS-CoV-2 nsp16/nsp10 enzyme complex methylates the 2′-OH of the first nucleotide of the viral mRNA, converting the Cap-0 to Cap-1, which helps the virus to evade immune surveillance in the host cell. Here, the authors present the crystal structure of SARS-CoV-2 nsp16/nsp10 with the bound Cap-1 RNA nucleotide product and a post-release SAH containing structure.
- Thiruselvam Viswanathan
- , Anurag Misra
- & Yogesh K. Gupta
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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 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 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 AccessComputationally-guided exchange of substrate selectivity motifs in a modular polyketide synthase acyltransferase
Engineering efforts have focused on acyltransferase (AT) domains of modular polyketide synthases (PKSs) to site-selectively modify the resulting polyketides, but critical AT residues involved in substrate selection have not been fully elucidated. Here, the authors use molecular dynamics to pinpoint mutations that impact AT domain selectivity and exchange structural motifs to obtain chimeric PKS modules with expanded substrate specificity.
- Edward Kalkreuter
- , Kyle S. Bingham
- & Gavin J. Williams
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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 AccessCreating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide
Nicotinamide cytosine dinucleotide is an NAD mimic that can be used to mediate redox reactions. Here the authors design an NCD synthetase for the intracellular production of NCD.
- Xueying Wang
- , Yanbin Feng
- & Zongbao K. Zhao
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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 AccessThe M-phase regulatory phosphatase PP2A-B55δ opposes protein kinase A on Arpp19 to initiate meiotic division
Mechanisms triggering meiotic divisions of oocytes remain unclear. Here, the authors report that meiosis resumption relies on the timely phosphorylation of Arpp19 protein at two distinct sites, which depends on two kinases (PKA and Gwl) and a single phosphatase (PP2A-B55δ).
- Tom Lemonnier
- , Enrico Maria Daldello
- & Aude Dupré
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Article
| Open AccessStructural and functional insights into esterase-mediated macrolide resistance
Erythromycin esterases (Eres) cleave the macrolactone ring of macrolides, a class of widely used antibiotics. Structures of EreC, in silico flexible docking studies and previous mutagenesis data lead to the proposal of a detailed catalytic mechanism for the Ere family of enzymes.
- Michał Zieliński
- , Jaeok Park
- & Albert M. Berghuis
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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 AccessDiscovery of fungal surface NADases predominantly present in pathogenic species
Some bacterial pathogens release NADase enzymes into the host cell that deplete the host’s NAD+ pool, thereby causing rapid cell death. Here, Strømland et al. identify NADases on the surface of fungal spores, and show that the enzymes display unique biochemical and structural properties.
- Øyvind Strømland
- , Juha P. Kallio
- & Mathias Ziegler
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Article
| Open AccessPervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics
Connecting conformational dynamics and epistasis has so far been limited to a few proteins and a single fitness trait. Here, the authors provide evidence of positive epistasis on multiple catalytic traits in the evolution and dynamics of engineered cytochrome P450 monooxygenase, offering insights for in silico protein design.
- Carlos G. Acevedo-Rocha
- , Aitao Li
- & Manfred T. Reetz
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Article
| Open AccessInhibitory mechanism of reveromycin A at the tRNA binding site of a class I synthetase
Reveromycin A (RM-A) selectively inhibits eukaryotic cytoplasmic isoleucyltRNA synthetase (IleRS). Herein, the authors show that RM-A molecule occupies the tRNAIle binding site of Saccharomyces cerevisiae IleRS, and that RM-A cooperates with isoleucine or isoleucyl-adenylate for IleRS binding.
- Bingyi Chen
- , Siting Luo
- & Huihao Zhou
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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 AccessSystem-wide identification and prioritization of enzyme substrates by thermal analysis
The global identification of enzyme substrates is still challenging. Here, the authors develop a method based on proteome-wide thermal shift assays to discover enzyme substrates directly from cell lysates, identifying known and novel oxidoreductase, kinase and poly-(ADP-ribose) polymerase substrates.
- Amir Ata Saei
- , Christian M. Beusch
- & Roman A. Zubarev
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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 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 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 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 AccessActive-site loop variations adjust activity and selectivity of the cumene dioxygenase
Active-site loops are important for catalytic properties of enzymes, but challenging to engineer due to their high flexibility and diversity. Here, the authors identify and engineer hot-spots in the loops of cumene dioxygenase, obtain variants with changed activity, regio- and enantioselectivity, and present a Linker In Loop Insertion approach for loop modification.
- Peter M. Heinemann
- , Daniel Armbruster
- & Bernhard Hauer
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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 AccessCryo-EM structures of engineered active bc1-cbb3 type CIII2CIV super-complexes and electronic communication between the complexes
Respiratory chains generate the proton motive force used for ATP synthesis. Cryo-EM structures of functional respiratory CIII2CIV supercomplex and native CIII2 from Rhodobacter capsulatus provide insight into CIII2CIV assembly and respiratory electron transport pathways in Gram-negative bacteria.
- Stefan Steimle
- , Trevor van Eeuwen
- & Fevzi Daldal