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| Open AccessContractile injection systems facilitate sporogenic differentiation of Streptomyces davawensis through the action of a phage tapemeasure protein-related effector
Bacteria can use contractile injection systems, similar to viral tail structures, to deliver toxic proteins into other cells. Here, Nagakubo et al. identify a related system that modulates sporulation in multicellular Streptomyces bacteria.
- Toshiki Nagakubo
- , Tatsuya Nishiyama
- & Masanori Toyofuku
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Article
| Open AccessA rationally designed miniature of soluble methane monooxygenase enables rapid and high-yield methanol production in Escherichia coli
Soluble methane monooxygenase (sMMO) is a potentially value biocatalyst, but production of active recombinant sMMO is very challenging. Here the authors report the rational design and construction of a catalytically active miniature sMMO which enables high-yield production of methanol in E. coli.
- Yeonhwa Yu
- , Yongfan Shi
- & Jeewon Lee
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Article
| Open AccessCystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region
Here the authors use phage display to develop cystine-knot peptides that inhibit the trimeric serine protease HTRA1. Structural and biochemical characterisation uncovered binding of the peptides to a cryptic pocket that locked the active site in a noncompetent state.
- Yanjie Li
- , Yuehua Wei
- & Daniel Kirchhofer
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Article
| Open AccessThe 8-17 DNAzyme can operate in a single active structure regardless of metal ion cofactor
Here, the authors use solution-state NMR to characterize the structure of 8–17 DNAzyme, revealing that all metal cofactors induce the same DNAzyme fold in contrast to previous findings.
- Julia Wieruszewska
- , Aleksandra Pawłowicz
- & Witold Andrałojć
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Article
| Open AccessMycobacterial biotin synthases require an auxiliary protein to convert dethiobiotin into biotin
Lipid biosynthesis in the pathogen M. tuberculosis depends on biotin for posttranslational modification of key enzymes. Here, Qu et al. identify an auxiliary protein that is required by M. tuberculosis to synthesize biotin.
- Di Qu
- , Peng Ge
- & Dirk Schnappinger
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Article
| Open AccessStructure, mechanism, and evolution of the last step in vitamin C biosynthesis
Photosynthetic organisms, fungi, and animals contain distinct pathways for vitamin C biosynthesis, but the final biosynthetic step consistently involves an oxidation reaction catalysed by the aldonolactone oxidoreductases. Here, the authors investigate the origin and evolution of the diversified activities and substrate preferences featured by these enzymes using different methods and find evidence that they share a common ancestor.
- Alessandro Boverio
- , Neelam Jamil
- & Andrea Mattevi
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Article
| Open AccessAnalysis of early intermediate states of the nitrogenase reaction by regularization of EPR spectra
Here, the authors characterize selenium and sulphur incorporated FeMo cofactors of the catalytic MoFe protein component from Azotobacter vinelandii under turnover conditions using EPR.
- Lorenz Heidinger
- , Kathryn Perez
- & Erik Schleicher
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Article
| Open AccessMutational dissection of a hole hopping route in a lytic polysaccharide monooxygenase (LPMO)
Lytic polysaccharide monooxygenases (LPMOs) are mono copper enzymes with outstanding industrial applicability. Here, the authors investigate the “hole hopping” mechanism in a bacterial LPMO and show that a strictly conserved tryptophan is critical for radical formation and hole transference, as well as reveal a correlation between the efficiency of hole transference and enzyme performance under oxidative stress.
- Iván Ayuso-Fernández
- , Tom Z. Emrich-Mills
- & Vincent G. H. Eijsink
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Article
| Open AccessGenetic and functional diversity of β-N-acetylgalactosamine-targeting glycosidases expanded by deep-sea metagenome analysis
Four β-N-acetylgalactosaminidase gene families were identified via deep-sea metagenome analysis. The biochemical and structural characterization of the aforementioned enzymes revealed their functional diversity and monophyletic evolutionary history.
- Tomomi Sumida
- , Satoshi Hiraoka
- & Takuro Nunoura
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Article
| Open AccessMolecular basis of bacterial DSR2 anti-phage defense and viral immune evasion
The defense-associated sirtuin 2 (DSR2) system protects bacteria from phages by depleting NAD+. Here, authors elucidate the molecular mechanisms underlying DSR2 assembly, activation, and inhibition, providing important insights into bacterial anti-phage defense.
- Jiafeng Huang
- , Keli Zhu
- & Ang Gao
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Article
| Open AccessInterplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis
Enzymes involve structural flexibility in their function, but understanding enzyme catalysis as connected to protein motions is a major challenge. Here, the authors obtain energetic description of C-H activation in nicotinamide coenzyme-dependent UDP-glucuronic acid C4 epimerase based on temperature kinetic studies and isotope effect measurements.
- Christian Rapp
- , Annika Borg
- & Bernd Nidetzky
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Article
| Open AccessPlatform-directed allostery and quaternary structure dynamics of SAMHD1 catalysis
SAMHD1 is a regulator of dNTP homeostasis and an HIV restriction factor. The authors use time-resolved cryo-EM to visualise dynamic conformational changes that drive the catalytic cycle and allosteric regulation of this multi-subunit enzyme.
- Oliver J. Acton
- , Devon Sheppard
- & Ian A. Taylor
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Article
| Open AccessNucleoside Phosphorylases make N7-xanthosine
Nucleoside-processing enzymes exhibit strict regioselectivity for glycosylation of purine nucleobases at N9. Here, the authors report an exception and show that wild type nucleoside phosphorylases also furnish N7-xanthosine, a non-native ribosylation regioisomer of xanthosine.
- Sarah Westarp
- , Felix Brandt
- & Felix Kaspar
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Article
| Open AccessCyclodipeptide oxidase is an enzyme filament
Many cyclic dipeptide natural products can be modified by cyclodipeptide oxidase enzymes. Here, the authors report the structural characterization of the cyclodipeptide oxidase AlbAB and show that it assembles into heterooligomeric enzyme filaments.
- Michael P. Andreas
- & Tobias W. Giessen
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Article
| Open AccessTyrosine phosphorylation of CARM1 promotes its enzymatic activity and alters its target specificity
Coactivator-associated arginine methyltransferase 1 (CARM1) is an important target in hematologic malignancies. In this work, the authors show that the hyperactivation of Janus kinase 2 (JAK2) by the V617F mutation phosphorylates CARM1 which regulates its methyltransferase activity and alters its target specificity.
- Hidehiro Itonaga
- , Adnan K. Mookhtiar
- & Stephen D. Nimer
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Article
| Open AccessStructural insights into the transporting and catalyzing mechanism of DltB in LTA D-alanylation
Here, the authors structurally and functionally characterise DltB, a member of the Membrane-Bound O-AcylTransferase (MBOAT) superfamily responsible for D-alanine incorporation in the cell wall of gram-positive bacteria.
- Pingfeng Zhang
- & Zheng Liu
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Article
| Open AccessThe genetic landscape of a metabolic interaction
Reynolds and colleagues examine a biochemically-mediated epistatic interaction between metabolic enzymes involved in folate metabolism and show that biochemical coupling shapes the range of enzyme activities sufficient to rescue cell growth.
- Thuy N. Nguyen
- , Christine Ingle
- & Kimberly A. Reynolds
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Article
| Open AccessAn enzymatic continuous-flow reactor based on a pore-size matching nano- and isoporous block copolymer membrane
Continuous-flow biocatalysis with immobilized enzymes is a sustainable route for chemical synthesis, but inadequate biocatalytic efficiency caused by non-productive enzyme immobilization or enzyme-carrier mismatches presents a challenge for its application. Here, the authors report an approach for the fabrication of a high-performance enzymatic continuous-flow reactor via integrating scalable isoporous block copolymer membranes as carriers with an oriented one-step enzyme immobilization via a genetically fused material binding peptide.
- Zhenzhen Zhang
- , Liang Gao
- & Volker Abetz
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Article
| Open AccessRemote loop evolution reveals a complex biological function for chitinase enzymes beyond the active site
Loop regions play a key role in protein evolution. Herein the authors demonstrate how GH19 chitinase acquired additional antifungal activity by introducing remote loops, without compromising its original function. This work offers an innovative approach to expand enzyme function.
- Dan Kozome
- , Adnan Sljoka
- & Paola Laurino
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Article
| Open AccessDynamic inter-domain transformations mediate the allosteric regulation of human 5, 10-methylenetetrahydrofolate reductase
Here the authors present the cryo-EM structure of active and inhibited human MTHFR, revealing a dynamic inhibitory mechanism dependent on dual SAM binding. The resulting closed conformation features an autoinhibitory element effectively blocking enzymatic activity.
- Linnea K. M. Blomgren
- , Melanie Huber
- & Thomas J. McCorvie
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Article
| Open AccessArchitecture and regulation of filamentous human cystathionine beta-synthase
Cystathionine beta-synthase is a conserved essential enzyme of one-carbon metabolism. Here, the authors show that the enzyme oligomerises to form filaments that undergo conformational and morphological changes in response to its activator S-adenosyl-L-methionine, the global methyl donor.
- Thomas J. McCorvie
- , Douglas Adamoski
- & Wyatt W. Yue
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Article
| Open AccessPARP2 promotes Break Induced Replication-mediated telomere fragility in response to replication stress
Here the authors show that PARP2 drives telomere fragility by orchestrating the Break-induced replication (BIR) pathway. This promotes DNA end resection and DNA synthesis via the regulation of POLD3.
- Daniela Muoio
- , Natalie Laspata
- & Elise Fouquerel
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Article
| Open AccessStructural basis for phage-mediated activation and repression of bacterial DSR2 anti-phage defense system
The bacterial DSR2 defense system counters phage invasion by depleting NAD+. Here, Zhang et al. reveal molecular mechanisms underlying phage-mediated activation and repression of DSR2, enhancing our understanding of the bacterial-phage arms race.
- Jun-Tao Zhang
- , Xiao-Yu Liu
- & Ning Jia
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Article
| Open AccessIdentifying a key spot for electron mediator-interaction to tailor CO dehydrogenase’s affinity
Carbon monoxide dehydrogenases (CODH) employ artificial electron mediators like viologens for biocatalysis, but little is known about the interaction between the mediators and the enzyme. Here, the authors discover the critical site for viologen interactions at the D-cluster of Carboxydothermus hydrogenoformans CODH2 via alanine mutations and crystallography, and report variants with increased ethyl viologen affinity.
- Suk Min Kim
- , Sung Heuck Kang
- & Yong Hwan Kim
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Article
| Open AccessArchitecture and activation of human muscle phosphorylase kinase
High-resolution cryo-EM study of human muscle phosphorylase kinase reveals its complex structure and how calcium ions activate it, offering insights into glycogen metabolism and kinase regulation.
- Xiaoke Yang
- , Mingqi Zhu
- & Junyu Xiao
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Article
| Open AccessSolar-driven sugar production directly from CO2 via a customizable electrocatalytic–biocatalytic flow system
Solar-driven artificial food synthesis from CO2 provides an approach to overcome the limitations of natural photosynthesis, but it is very challenging. Here, the authors report a hybrid electrocatalytic-biocatalytic flow system, coupling photovoltaics-powered electrocatalysis (CO2 to formate) with a five enzyme cascade platform (formate to sugar), which achieves conversion of CO2 to C6 sugar (L-sorbose) with a solar-to-food energy conversion efficiency of 3.5%.
- Guangyu Liu
- , Yuan Zhong
- & Yujie Xiong
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Article
| Open AccessThe evolutionary origin of naturally occurring intermolecular Diels-Alderases from Morus alba
Diels-Alderases (DAs), enzymes catalyzing [4 + 2] cycloaddition reactions, are of high interest, but insights into their evolution are lacking. Here, the authors investigate the evolutionary origins of the intermolecular DAs in the biosynthesis of Moraceae plant-derived Diels-Alder-type secondary metabolites, suggesting they evolved from an ancestor functioning as a flavin adenine dinucleotide-dependent oxidocyclase.
- Qi Ding
- , Nianxin Guo
- & Xiaoguang Lei
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Article
| Open AccessLegionella metaeffector MavL reverses ubiquitin ADP-ribosylation via a conserved arginine-specific macrodomain
The pathogen Legionella pneumophila mediates NAD+-dependent ubiquitination pathways upon infection. Here, the authors show the Legionella effector MavL reverses ubiquitin ADP-ribosylation to regulate these pathways. MavL represents a new macrodomain class specific for reversal of arginine ADP-ribosylation with distinct ADP-ribose binding features.
- Zhengrui Zhang
- , Jiaqi Fu
- & Chittaranjan Das
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Article
| Open AccessSupra-biological performance of immobilized enzymes enabled by chaperone-like specific non-covalent interactions
Designing complex synthetic materials for enzyme immobilization could unlock the utility of biocatalysis in extreme environments. Here, the authors report on random copolymer brushes as dynamic immobilization supports that enable supra-biological catalytic performance of immobilized enzymes.
- Héctor Sánchez-Morán
- , Joel L. Kaar
- & Daniel K. Schwartz
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Article
| Open AccessA three-level regulatory mechanism of the aldo-keto reductase subfamily AKR12D
Here, the authors characterise an aldo-keto reductase AKRtyl, which belongs to a previously unidentified subfamily AKR12D. They uncover a complex mechanism of allosteric regulation that is mediated by 3 distinct states.
- Zhihong Xiao
- , Jinyin Zha
- & Shaobo Dai
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Article
| Open AccessDJ-1 protects proteins from acylation by catalyzing the hydrolysis of highly reactive cyclic 3-phosphoglyceric anhydride
Human protein DJ-1 displays neuroprotective properties. Here, the authors demonstrate that DJ-1 hydrolyzes cyclic 3-phosphoglyceric anhydride (cPGA), thereby protecting proteins from acylation by this highly reactive metabolite spontaneously forming in glycolysis.
- Aizhan Akhmadi
- , Adilkhan Yeskendir
- & Darkhan Utepbergenov
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Article
| Open AccessA non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme
The authors previously showed that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita Baylis-Hillman (MBH) reaction. Here, they report another efficient MBHase that employs a non-canonical Nδ-methylhistidine nucleophile paired with a catalytic glutamate, providing an alternative mechanistic solution for MBH catalysis.
- Amy E. Hutton
- , Jake Foster
- & Anthony P. Green
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Article
| Open AccessUnderstanding activity-stability tradeoffs in biocatalysts by enzyme proximity sequencing
Understanding the complex relationships between enzyme sequence, folding stability and catalytic activity is essential for applications, but current technologies cannot simultaneously resolve both stability and activity phenotypes and couple these to gene sequences at large scale. Here, the authors report Enzyme Proximity Sequencing (EP-Seq), a deep mutational scanning method to assay both expression level and catalytic activity of thousands of oxidoreductase variants from a cellular pool in a single experiment.
- Rosario Vanella
- , Christoph Küng
- & Michael A. Nash
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Article
| Open AccessStructure-function analysis of the cyclic β-1,2-glucan synthase from Agrobacterium tumefaciens
Here, the authors present the structure of cyclic β-1,2-glucan synthase from Agrobacterium tumefaciens, revealing a distinct mechanism that uses a tyrosine-linked oligosaccharide intermediate in cycles of polymerization and processing of the glucan chain.
- Jaroslaw Sedzicki
- , Dongchun Ni
- & Christoph Dehio
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Article
| Open AccessDeciphering the mechanism of glutaredoxin-catalyzed roGFP2 redox sensing reveals a ternary complex with glutathione for protein disulfide reduction
Fusion proteins between roGFP2 and glutaredoxins are used for intracellular redox measurements. Here, the authors determined all rate constants of the reaction cycle for roGFP2 measurements and identified an alternative glutaredoxin mechanism.
- Fabian Geissel
- , Lukas Lang
- & Marcel Deponte
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Article
| Open AccessCreation of a point-of-care therapeutics sensor using protein engineering, electrochemical sensing and electronic integration
Low-cost point-of-care sensors are vital for precision medicine. Here, the authors have repurposed a glucometer for breast cancer therapeutic detection capable of sensing tamoxifen in human blood, utilizing blood glucose to power and amplify the therapeutic signals
- Rong Cai
- , Chiagoziem Ngwadom
- & Caroline M. Ajo-Franklin
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Article
| Open AccessNAAA-regulated lipid signaling in monocytes controls the induction of hyperalgesic priming in mice
Circulating monocytes contribute to the transition to pain chronicity but the molecular events that cause their deployment are still unclear. Using a mouse model of hyperalgesic priming, here the authors show that blood monocytes contribute to the emergence of chronic pain via a mechanism that requires a transient disruption of NAAA-regulated lipid signaling.
- Yannick Fotio
- , Alex Mabou Tagne
- & Daniele Piomelli
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Article
| Open AccessSpatiotemporal and direct capturing global substrates of lysine-modifying enzymes in living cells
Here the authors report a strategy to directly capture substrates of lysine-modifying enzymes via post-translational modification (PTM)-acceptor residue crosslinking in living cells, enabling global profiling of substrates of PTM-enzymes and validation of PTM-sites in a straightforward manner.
- Hao Hu
- , Wei Hu
- & Xiao-Hua Chen
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Article
| Open AccessStructural and mechanistic characterization of bifunctional heparan sulfate N-deacetylase-N-sulfotransferase 1
Heparan sulfate biosynthesis is a complex process involving multiple reactions that extend and modify the polysaccharide. Here, the authors resolve structures of NDST1, responsible for the critical N-sulfoglucosamine modification of heparan sulfate.
- Courtney J. Mycroft-West
- , Sahar Abdelkarim
- & Liang Wu
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Article
| Open AccessA distinctive family of L,D-transpeptidases catalyzing L-Ala-mDAP crosslinks in Alpha- and Betaproteobacteria
The cell-wall peptidoglycan in model bacteria typically includes 4,3- and 3,3-crosslinks, catalysed by DD- and LD-transpeptidases, respectively. Here, the authors identify and characterise the activity and structure of an LD-transpeptidase that generates a new type of crosslink (1,3).
- Akbar Espaillat
- , Laura Alvarez
- & Felipe Cava
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Article
| Open AccessPoly-γ-glutamylation of biomolecules
Poly-γ-glutamate tails are a distinctive feature of folate and F420 cofactors, but it was unclear how these tails elongate while maintaining substrate specificity. Here, the authors discover that folylpolyglutamate synthase and γ-glutamyl ligase enzymes add successive L-glutamates to the termini of the growing γ-glutamyl chain in a processive mechanism.
- Ghader Bashiri
- , Esther M. M. Bulloch
- & Christopher J. Squire
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Article
| Open AccessNatural diversity screening, assay development, and characterization of nylon-6 enzymatic depolymerization
Polyamides (PAs) or nylons are types of plastics with wide applications, but due to their accumulation in the environment, strategies for their deconstruction are of interest. Here, the authors screen 40 potential nylon-hydrolyzing enzymes (nylonases) using a mass spectrometry-based approach and identify a thermostabilized N-terminal nucleophile hydrolase as the most promising for further development, as well as crucial targets for progressing PA6 enzymatic depolymerization.
- Elizabeth L. Bell
- , Gloria Rosetto
- & Gregg T. Beckham
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Article
| Open AccessUsing the heme peroxidase APEX2 to probe intracellular H2O2 flux and diffusion
Previous genetically encoded H2O2 probes are based on reversible thiol oxidation. Here, a heme peroxidase is introduced as a thiol-independent H2O2 probe. APEX2 converts H2O2 into fluorescent or luminescent signals, allowing its quantification.
- Mohammad Eid
- , Uladzimir Barayeu
- & Tobias P. Dick
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Article
| Open AccessA universal metabolite repair enzyme removes a strong inhibitor of the TCA cycle
Succinate dehydrogenase converts malate to enoloxaloacetate, a metabolically inactive and inhibitory side product of the TCA cycle. Here, Zmuda et al. describe a conserved metabolite damage repair enzyme that can remove enol-oxaloacetate and is critical for efficient aerobic respiration.
- Anthony J. Zmuda
- , Xiaojun Kang
- & Thomas D. Niehaus
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Article
| Open AccessA renal clearable fluorogenic probe for in vivo β-galactosidase activity detection during aging and senolysis
In vivo detection of cell senescence remains a challenge in aging research. This work introduces a novel fluorogenic probe for β-Gal activity that is excreted in urine, providing a simple diagnosis method to estimate the systemic load of senescent cells during aging and senolytic interventions.
- Sara Rojas-Vázquez
- , Beatriz Lozano-Torres
- & Ramón Martínez-Máñez
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Article
| Open AccessMultiple redox switches of the SARS-CoV-2 main protease in vitro provide opportunities for drug design
Here the authors demonstrate that the SARS-CoV-2 main protease (Mpro) is subject to redox regulation in vitro, reversibly switching between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues.
- Lisa-Marie Funk
- , Gereon Poschmann
- & Kai Tittmann
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Article
| Open AccessThe polyketide to fatty acid transition in the evolution of animal lipid metabolism
Much is still unknown of the evolution of animal metabolic enzymes. This study describes a new enzyme family bridging the production of polyketides and membrane lipids. This expands the known biochemical repertoire of animals for making ecologically and biomedically important natural products.
- Zhenjian Lin
- , Feng Li
- & Eric W. Schmidt
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Article
| Open AccessReprogramming biocatalytic futile cycles through computational engineering of stereochemical promiscuity to create an amine racemase
Racemization of chiral amines poses a challenge, making dynamic kinetic resolution inaccessible for industrial applications. Here, the authors demonstrate in silico engineering of an amine transaminase to create a new-to-nature amine racemase.
- Sang-Woo Han
- , Youngho Jang
- & Jong-Shik Shin
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Article
| Open AccessPervasive epistasis exposes intramolecular networks in adaptive enzyme evolution
Here, the authors perform statistical analyses to demonstrate that epistasis is highly pervasive in adaptive evolutionary trajectories of enzymes. Using epistatic data, they expose higher-order rewiring of intramolecular amino acid networks.
- Karol Buda
- , Charlotte M. Miton
- & Nobuhiko Tokuriki