Enzymes

  • Article
    | Open Access

    Microbial DNA glycosylases associated with the biosynthesis of DNA-damaging antibiotics have evolved self-resistance for their cognate natural products. Here, the authors provide evidence that cellular self-resistance is enabled by reduced affinity of the glycosylases for the excision products of the corresponding DNA lesions.

    • Elwood A. Mullins
    • , Jonathan Dorival
    •  & Brandt F. Eichman
  • Article
    | Open Access

    AGPATs (1-acylglycerol-3-phosphate O-acyltransferases) catalyze the acylation of lysophosphatidic acid to form phosphatidic acid (PA), a key step in the synthesis of all glycerolipids. Here, the authors show that AGPAT2 and CDP-DAG synthases (CDS1 and CDS2) form functional complexes that promote further conversion of PA along the CDP-DAG pathway of phospholipid synthesis.

    • Hoi Yin Mak
    • , Qian Ouyang
    •  & Hongyuan Yang
  • Article
    | Open Access

    L-asparaginases catalyse the hydrolysis of L-asparagine to L-aspartic acid and ammonia. Here, the authors present high resolution crystal structures of Rhizobium etli L-asparaginase that contains a Zn2+ binding site without a catalytic role and discuss the catalytic mechanism of the enzyme.

    • Joanna I. Loch
    • , Barbara Imiolczyk
    •  & Mariusz Jaskolski
  • Article
    | Open Access

    The nucleotidyl cyclase toxin exoenzyme Y (ExoY), which is secreted by the human pathogens Pseudomonas aeruginosa and Vibrio vulnificus is activated by actin. Here, the authors present the cryo-EM structures of PaExoY bound to F-actin and VvExoY in complex with G-actin-profilin. These structures together with molecular dynamics simulations and enzymatic assays provide insights into the activation mechanism for both bacterial cyclase toxin families that interact with either F- or G-actin.

    • Alexander Belyy
    • , Felipe Merino
    •  & Stefan Raunser
  • Article
    | Open Access

    The modification of proteins with O-linked β-N-acetylglucosamine (OGlcNAc) plays roles in regulation of numerous cellular functions while incorrect O-GlcNAcylation patterns are linked to disease. Here, the authors report a cryo-EM structure of full-length O-GlcNAc transferase (OGT), the only enzyme responsible for O-GlcNAcylation.

    • Richard W. Meek
    • , James N. Blaza
    •  & Gideon J. Davies
  • Article
    | Open Access

    The human 2-oxoglutarate (2OG) oxygenases FIH and AspH are relevant drug targets. Here, the authors show that synthetic and naturally occurring 2OG derivatives can selectively modulate FIH and AspH activities, suggesting that these compounds may serve as a basis to develop 2OG oxygenase-targeting probes and drugs.

    • Yu Nakashima
    • , Lennart Brewitz
    •  & Christopher J. Schofield
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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