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| Open AccessAntibiotic hyper-resistance in a class I aminoacyl-tRNA synthetase with altered active site signature motif
Aminoacyl-tRNA synthetases translate the genetic code. These enzymes harbor signature catalytic motifs dating from their ancient ancestors. A natural variation of one of the stated motifs was discovered and linked to antibiotic hyper-resistance.
- A. Brkic
- , M. Leibundgut
- & I. Gruic-Sovulj
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Article
| Open AccessO-methyltransferase-like enzyme catalyzed diazo installation in polyketide biosynthesis
Diazo compounds, such as kinamycin, are rare bioactive natural products whose assembly has been extensively studied, but the formation of the diazo group is elusive. Here, the authors report O-methyltransferase-like protein, AlpH, which is responsible for the l-glutamylhydrazine incorporation in kinamycin biosynthesis.
- Yuchun Zhao
- , Xiangyang Liu
- & Ming Jiang
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Article
| Open AccessNext generation synthetic memory via intercepting recombinase function
Memory is a basic tenet of intelligent biological systems. Here the authors engineered a programmable and expandable iteration of recombinase-based synthetic memory (interception) that functions post-translation, resulting in faster recombination.
- Andrew E. Short
- , Dowan Kim
- & Corey J. Wilson
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Article
| Open AccessArchitecture of the Heme-translocating CcmABCD/E complex required for Cytochrome c maturation
The covalent linkage of hemes to cytochromes c requires a maturation machinery. Here, the authors provide mechanistic insights into how the heme translocase complex CcmABCD flops a heme group, driven by ATP hydrolysis, and delivers it to the chaperone CcmE.
- Lorena Ilcu
- , Lukas Denkhaus
- & Oliver Einsle
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Article
| Open AccessGlycerol 3-phosphate phosphatase/PGPH-2 counters metabolic stress and promotes healthy aging via a glycogen sensing-AMPK-HLH-30-autophagy axis in C. elegans
Activation of the glycerol shunt mimics calorie restriction, protects from glucotoxicity, and promotes healthy aging. Here, Possik et al. uncover the underlying mechanism and identify the AMPK-TFEB autophagy pathway as critical to the healthy aging phenotype.
- Elite Possik
- , Laura-Lee Klein
- & Marc Prentki
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Article
| Open AccessThermodynamic principle to enhance enzymatic activity using the substrate affinity
Currently, there is no well-defined strategy to increase the activity of enzymes. Here, the authors provide mathematical evidence that adjusting the Michaelis-Menten constant to the substrate concentration maximizes enzymatic activity.
- Hideshi Ooka
- , Yoko Chiba
- & Ryuhei Nakamura
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Article
| Open AccessStructural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex
Bacterial cell shape is dependent on the formation of the extracellular sugar polymer called peptidoglycan. Here the authors describe RodA-PBP2, the enzymatic core of the elongasome, which is the complex responsible peptidoglycan synthesis, and utilize an integrated approach to investigate the mechanism of peptidoglycan biosynthesis.
- Rie Nygaard
- , Chris L. B. Graham
- & Filippo Mancia
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Article
| Open AccessStructure and regulation of full-length human leucine-rich repeat kinase 1
Leucine-rich repeat kinase 1 (LRRK1) and its counterpart LRRK2 play crucial roles in regulating fundamental cellular processes. Here, the authors use cryo-EM to characterize the LRRK1 monomer and dimer, revealing interfaces that regulate kinase activity and structural differences to LRRK2.
- Riley D. Metcalfe
- , Juliana A. Martinez Fiesco
- & Ping Zhang
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Article
| Open AccessCatalytic site flexibility facilitates the substrate and catalytic promiscuity of Vibrio dual lipase/transferase
Vibrio dual lipases/transferases are virulence-related enzymes, with both substrate and catalytic promiscuity. Wang et al reveal their prominent structural flexibility, proposing a catalytic site tuning mechanism underlying enzyme promiscuity.
- Chongyang Wang
- , Changshui Liu
- & Qingjun Ma
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Article
| Open AccessCryo-EM structures of Uba7 reveal the molecular basis for ISG15 activation and E1-E2 thioester transfer
ISGylation plays a crucial role in the innate immune response and requires sequential activity of E1, E2, and E3 enzymes. Here, the authors present cyro-EM structures that reveal the molecular mechanisms underlying ISG15 activation by the E1 enzyme Uba7 and transfer to its cognate E2 enzyme UBE2L6.
- Mohammad Afsar
- , GuanQun Liu
- & Shaun K. Olsen
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Article
| Open AccessStructure, catalysis, chitin transport, and selective inhibition of chitin synthase
Chitin, the second most abundant natural polysaccharide in nature, is synthesized by chitin synthases, which are recognized as targets for antifungal and anti-insect drugs. Here the authors determine cryo-EM structures of the chitin synthase, which reveal its activation, catalytic and inhibitory mechanisms
- Dan-Dan Chen
- , Zhao-Bin Wang
- & Lin Bai
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Article
| Open AccessStructure of lasso peptide epimerase MslH reveals metal-dependent acid/base catalytic mechanism
MslH, encoded in the MS-271 biosynthetic gene cluster, catalyzes the epimerization at the Cα center of the MslA C-terminal Trp21, however, the detailed catalytic process was unknown. Here, the authors report MslH is a metallo-dependent peptide epimerase with a calcineurin-like fold.
- Yu Nakashima
- , Atsushi Kawakami
- & Hiroyuki Morita
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Article
| Open AccessMolecular basis of β-lactam antibiotic resistance of ESKAPE bacterium E. faecium Penicillin Binding Protein PBP5
Penicillin Binding Proteins (PBPs) are the main targets of β-lactam antibiotics. Here the authors use NMR spectroscopy, crystallography and microbiology to define the dynamics of E. faecium PBP5 in solution and show that increased acyl-enzyme hydrolysis correlates with increased resistance.
- Yamanappa Hunashal
- , Ganesan Senthil Kumar
- & Wolfgang Peti
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Article
| Open AccessMechanism of ATP hydrolysis dependent rotation of bacterial ATP synthase
The ATP synthase FoF1 undergoes rotation in discrete 120° steps. Using cryo-EM analysis, the authors characterise intermediate structures within these 120° steps at 81°, 83°, 91°, and 101°. This shows that FoF1 undergoes a total of 15 steps in a 360° rotation, exhibiting multiple discreet movements per full rotation as opposed to one fluid motion.
- Atsuki Nakano
- , Jun-ichi Kishikawa
- & Ken Yokoyama
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Article
| Open AccessStructures of a FtsZ single protofilament and a double-helical tube in complex with a monobody
The cryoEM structures of a single protofilament of FtsZ from Klebsiella pneumoniae (KpFtsZ) in a polymerization-preferred conformation are presented and of a double-helical tube of the FtsZ–monobody complex that shows two parallel protofilaments.
- Junso Fujita
- , Hiroshi Amesaka
- & Hiroyoshi Matsumura
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Article
| Open AccessMolecular insights into the catalytic promiscuity of a bacterial diterpene synthase
Diterpene synthase VenA catalyses the synthesis of venezuelaene A with a unique 5-5-6-7 tetracyclic skeleton from geranylgeranyl pyrophosphate. Here, the authors report crystal structures of apo- and holo-VenA, provide mechanistic insights into its substrate selectivity and promiscuity, and engineer VenA into a sesterterpene synthase.
- Zhong Li
- , Lilan Zhang
- & Shengying Li
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Article
| Open AccessStructural insight into the human SID1 transmembrane family member 2 reveals its lipid hydrolytic activity
The SID-1 family is a putative channel/transporter for transporting nucleic acids. Here, Qian et al. report the dimeric structure of human SIDT2 suggesting that it may act as a transporter but not a channel. In addition, it has a ceramidase activity.
- Dandan Qian
- , Ye Cong
- & Deshun Gong
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Article
| Open AccessMechanistic studies of a lipase unveil effect of pH on hydrolysis products of small PET modules
Plastic-degrading enzymes can be utilized for plastic recycling. Here, QM/MM molecular dynamics and experimental Michaelis–Menten kinetics provide insight into PETase/MHTase activities of the lipase B from Candida antartica.
- Katarzyna Świderek
- , Susana Velasco-Lozano
- & Vicent Moliner
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Article
| Open AccessAllosteric activation of cell wall synthesis during bacterial growth
Coordination of cell wall assembly is critical for bacterial morphology and survival. Here, the authors show that activation of cell wall synthesis by the Rod complex is regulated by the structural dynamics of RodA-PBP2.
- Irina Shlosman
- , Elayne M. Fivenson
- & Joseph J. Loparo
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Article
| Open AccessA multicentric consortium study demonstrates that dimethylarginine dimethylaminohydrolase 2 is not a dimethylarginine dimethylaminohydrolase
While dimethylarginine dimethylaminohydrolase 1 (DDAH1) is known to metabolize the endogenous inhibitor of nitric oxide synthases, asymmetric dimethylarginine (ADMA), the function of DDAH2 has remained controversial. Here, the authors present several lines of evidence that DDAH2 does not hydrolyze ADMA.
- Vinitha N. Ragavan
- , Pramod C. Nair
- & Roman N. Rodionov
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Article
| Open AccessA 2.2 Å cryoEM structure of a quinol-dependent NO Reductase shows close similarity to respiratory oxidases
Quinol-dependent nitric oxide reductases, unique to bacteria, are considered members of respiratory heme copper oxidases. A 2.2 Å cryoEM structure of qNOR is reported shedding light on key aspects of enzyme mechanism including quinol binding and pathways for electron, substrate, and proton transport.
- Alex J. Flynn
- , Svetlana V. Antonyuk
- & S. Samar Hasnain
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Article
| Open AccessDiscovery and biosynthesis of tricyclic copper-binding ribosomal peptides containing histidine-to-butyrine crosslinks
Cyclic peptides are important bioactive compounds and drugs, synthesised by enzymatic side-chain macrocyclization of ribosomal peptides, which rarely involves histidine residues. Here, the authors report the discovery and biosynthesis of tricyclic lanthipeptide noursin, constrained by a tri amino acid labionin crosslink and histidine-to-butyrine crosslink, which is important for copper binding of noursin.
- Yuqing Li
- , Yeying Ma
- & Huan Wang
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Article
| Open AccessNucleotide exchange is sufficient for Hsp90 functions in vivo
A complete understanding of the role of ATP hydrolysis in Hsp90 function is elusive. Here, the authors show that ATP hydrolysis, but not binding, is dispensable for essential or specialized Hsp90 functions in vivo, shedding new light on this mystery.
- Michael Reidy
- , Kevin Garzillo
- & Daniel C. Masison
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Article
| Open AccessMillisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography
The authors introduce the spitrobot, a crystal plunger, enabling cryo-trapping with millisecond time-resolution via the liquid application method (LAMA). Ligand binding and reaction intermediates are demonstrated in three different enzymes.
- Pedram Mehrabi
- , Sihyun Sung
- & Eike C. Schulz
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Article
| Open AccessComputational design and molecular dynamics simulations suggest the mode of substrate binding in ceramide synthases
Membrane proteins are involved in many critical cellular pathways. Here, authors use a combination of structural predictions, an algorithm for stabilizing membrane proteins, and molecular dynamics to reveal a putative mechanism for the action of ceramide synthases.
- Iris D. Zelnik
- , Beatriz Mestre
- & Anthony H. Futerman
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Article
| Open AccessAllosteric role of the citrate synthase homology domain of ATP citrate lyase
ATP citrate lyase (ACLY) is the main nucleocytosolic source of acetyl-CoA and the enzyme contains citrate synthase homology (CSH) and acyl-CoA synthetase homology (ASH) domains. Here, the authors report data on an ACLY mutant that supports an allosteric role for the CSH domain in ACLY catalysis.
- Xuepeng Wei
- , Kollin Schultz
- & Ronen Marmorstein
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Article
| Open AccessDephosphocholination by Legionella effector Lem3 functions through remodelling of the switch II region of Rab1b
During infection, the Legionella effector Lem3 removes a phosphocholine moiety from the human protein Rab1. Here, the authors present the crystal structure of the stabilised Lem3:Rab1b complex, revealing the catalytic mechanism and substrate recognition of PPM phosphatases shaped Lem3.
- Marietta S. Kaspers
- , Vivian Pogenberg
- & Aymelt Itzen
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Article
| Open AccessThe structural basis of tRNA recognition by arginyl-tRNA-protein transferase
ATE1 is a highly specific enzyme hijacking tRNA from ribosomal pathways to install an arginine onto proteins as a post-translational modification. Here, the authors describe the structures of yeast ATE1 with or without its tRNA cofactor. ATE1 recognizes and selects tRNA in a unique mechanism.
- Thilini Abeywansha
- , Wei Huang
- & Yi Zhang
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Article
| Open AccessTrim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates
TRIM21 mediates intracellular antibody immunity and is exploited for targeted protein degradation using Trim-Away technology. Here, the authors dissect the ubiquitination requirements for Trim-Away, providing an explanation for how TRIM21 can target diverse substrates for degradation.
- Leo Kiss
- , Tyler Rhinesmith
- & Leo C. James
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Article
| Open AccessNucleotide binding halts diffusion of the eukaryotic replicative helicase during activation
Here the authors examine fully reconstituted and activated CMG helicases at the single-molecule level. They observe unidirectional translocation by CMG in ATP and diffusive motion by CMG without ATP, where the latter can be halted by nucleotide binding.
- Daniel Ramírez Montero
- , Humberto Sánchez
- & Nynke H. Dekker
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Article
| Open AccessExtended DNA threading through a dual-engine motor module of the activating signal co-integrator 1 complex
ASCC3 is a multi-functional helicase that contains two consecutive Ski2-like helicase units. Here, the authors show that ASCC3 can unwind DNA by threading one strand of a substrate duplex through both helicase units, supported by the TRIP4 protein.
- Junqiao Jia
- , Tarek Hilal
- & Markus C. Wahl
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Article
| Open AccessMolecular mechanism on forcible ejection of ATPase inhibitory factor 1 from mitochondrial ATP synthase
IF1 is a natural inhibitor of mitochondrial FoF1-ATP synthase, which blocks catalysis and rotation of the F1 motor. Here, the authors show the rotational-direction-dependence of activation from IF1 inhibition, with IF1 being readily dissociated when F1 rotates to the clockwise direction.
- Ryohei Kobayashi
- , Hiroshi Ueno
- & Hiroyuki Noji
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Article
| Open AccessUnnatural activities and mechanistic insights of cytochrome P450 PikC gained from site-specific mutagenesis by non-canonical amino acids
The 20 proteinogenic amino acids cannot always satisfy the requirements of protein engineering. Here, the authors practice semi-rational non-canonical amino acid mutagenesis for a cytochrome P450 enzyme, leading to unnatural activities and significant mechanistic insights.
- Yunjun Pan
- , Guobang Li
- & Shengying Li
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Article
| Open AccessC–N bond formation by a polyketide synthase
Polyketide synthases (PKSs) usually form C–C bonds in natural products biosynthesis. Here, the authors present cryo-EM structures of a PKS in complex with products, which provides insight into the mechanism of the unexpected C–N bond formation.
- Jialiang Wang
- , Xiaojie Wang
- & Jingdan Liang
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Article
| Open AccessrRNA methylation by Spb1 regulates the GTPase activity of Nog2 during 60S ribosomal subunit assembly
Regulation of 60S biogenesis remains poorly understood. Using cryo-EM, the authors show that failure of Spb1 to methylate the A-loop nucleotide G2922 prematurely activates the GTPase Nog2, suggesting that Spb1 and Nog2 form a kinetic checkpoint during ribosome maturation.
- Kamil Sekulski
- , Victor Emmanuel Cruz
- & Jan P. Erzberger
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Article
| Open AccessStructural consequences of turnover-induced homocitrate loss in nitrogenase
Biological nitrogen fixation is achieved by nitrogenase, but the mechanism remains enigmatic. Here, the authors report high resolution single particle cryoEM structures of homocitrate-compromised MoFe-proteins and unveil a new binding partner.
- Rebeccah A. Warmack
- , Ailiena O. Maggiolo
- & Douglas C. Rees
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Article
| Open AccessStructural remodelling of the carbon–phosphorus lyase machinery by a dual ABC ATPase
Here, authors analyse the structural organisation of the large carbon-phosphorus lyase enzyme from bacteria using electron microscopy and discover that it contains two ATP-binding cassette dimers of PhnK and PhnL and opens upon ATP hydrolysis.
- Søren K. Amstrup
- , Sui Ching Ong
- & Ditlev E. Brodersen
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Article
| Open AccessSmall molecule inhibitors of 15-PGDH exploit a physiologic induced-fit closing system
Inhibition of 15-prostaglandin dehydrogenase (15-PGDH) is a promising therapeutic target for regenerative medicine. We report the structure of 15-PGDH in complex with two different inhibitors. Unexpectedly, access to the binding pocket is regulated by a dynamic “lid” of the enzyme.
- Wei Huang
- , Hongyun Li
- & Derek J. Taylor
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Article
| Open AccessInsights into azalomycin F assembly-line contribute to evolution-guided polyketide synthase engineering and identification of intermodular recognition
Intermodular recognition in polyketide synthase (PKS) is a key prerequisite for catalysis and assembly-line engineering. Here, the authors present a specific domain interaction between modules and an evolution-oriented strategy for PKS engineering derived from the enoylreduction module of azalomycin F.
- Guifa Zhai
- , Yan Zhu
- & Yuhui Sun
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Article
| Open AccessAn engineered variant of MECR reductase reveals indispensability of long-chain acyl-ACPs for mitochondrial respiration
Mitochondrial fatty acid synthesis (mtFAS) generates the precursor for lipoic acid synthesis, but the role of longer fatty acid products has remained unclear. Here, the authors generated an engineered variant of human 2E-enoyl-ACP reductase (MECR) of mtFAS to study the role of long chain fatty acids.
- M. Tanvir Rahman
- , M. Kristian Koski
- & Kaija J. Autio
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Article
| Open AccessEmergence of a proton exchange-based isomerization and lactonization mechanism in the plant coumarin synthase COSY
Plant biosynthetic enzymes rapidly evolve to catalyze specialized reactions. Here, the authors present the crystal structure and mechanism of COSY, the enzyme involved in coumarin biosynthesis of the BAHD-acyltransferase family that catalyzes an intramolecular acyl transfer reaction through a proton exchange mechanism.
- Colin Y. Kim
- , Andrew J. Mitchell
- & Jing-Ke Weng
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Article
| Open AccessStructural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization
Biological processes can be regulated via oligomerization of macromolecules into high-order symmetric structures. Here, authors reported high-order structure of RNA polymerase and its role in regulation of gene expression in pathogenic bacterium.
- Zakia Morichaud
- , Stefano Trapani
- & Konstantin Brodolin
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Article
| Open AccessStructural analysis of an anthrol reductase inspires enantioselective synthesis of enantiopure hydroxycycloketones and β-halohydrins
Asymmetric reduction of prochiral ketones is challenging. Here, the authors identify and solve the structure of anthrol reductase CbAR, whose variant H162F can convert 1,3-cyclodiketones and α-haloacetophenones to the corresponding chiral alcohols.
- Xiaodong Hou
- , Huibin Xu
- & Yijian Rao
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Article
| Open AccessInsights into the mechanism of phospholipid hydrolysis by plant non-specific phospholipase C
Non-specific phospholipase C (NPC) is a plant-specific phospholipid-hydrolyzing enzyme. Here, the authors define the molecular basis of how Arabidopsis NPC works, and provide new mechanistic insights into the members of phospholipase family.
- Ruyi Fan
- , Fen Zhao
- & Zhu Liu
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Article
| Open AccessThe unifying catalytic mechanism of the RING-between-RING E3 ubiquitin ligase family
RBR E3 ubiquitin ligases utilise a 2-step catalytic mechanism previously defined for only few of the RBR family members. Here, the authors examine the poorly studied RBRs HOIL-1 and RNF216 to define general principles of RBR catalysis and regulation and identify specific functional differences.
- Xiangyi S. Wang
- , Thomas R. Cotton
- & Bernhard C. Lechtenberg
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Article
| Open AccessA conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1
SARM1 is a key player in axon degeneration. Here, the authors generate a nanobody, which specifically recognizes the NMN-bound state of SARM1 and helps resolve the SARM1 structure in an intermediate state of activation.
- Yun Nan Hou
- , Yang Cai
- & Yong Juan Zhao
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Article
| Open AccessThe UFM1 system regulates ER-phagy through the ufmylation of CYB5R3
The UFM1 system, a ubiquitin-like conjugation system is crucial for endoplasmic reticulum (ER) homeostasis. Here, authors found that CYB5R3 is covalently conjugated with UFM1, which becomes a signal for ER-phagy, a selective autophagy of ER.
- Ryosuke Ishimura
- , Afnan H. El-Gowily
- & Masaaki Komatsu
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Article
| Open AccessMechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing
In this work, Blair and co-authors used cryo-EM and in vitro assays to show that human Ligase 1 (Lig1) and Flap Endonuclease 1 (FEN1) form a toolbelt with the sliding clamp PCNA coordinating the sealing of Okazaki fragments.
- Kerry Blair
- , Muhammad Tehseen
- & Alfredo De Biasio
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Article
| Open AccessRational enzyme design for enabling biocatalytic Baldwin cyclization and asymmetric synthesis of chiral heterocycles
Chiral heterocyclic compounds are privileged structures in medicinal chemistry. Here, the authors report an in silico strategy for the enzymatic synthesis of pharmaceutically significant chiral N- and O-heterocycles via Baldwin cyclization of hydroxy- and amino-substituted epoxides and oxetanes using epoxide hydrolase mutants.
- Jun-Kuan Li
- , Ge Qu
- & Zhoutong Sun