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Pseudaminic acid is a non-mammalian sugar found in the surface glycoconjugates of many bacteria, including several human pathogens, and is a virulence factor thought to facilitate immune evasion. The final step in the biosynthesis of the nucleotide activated form of the sugar, CMP-Pse5Ac7Ac is performed by a CMP-Pse5Ac7Ac synthetase (PseF). Here we present the biochemical and structural characterization of PseF from Aeromonas caviae (AcPseF), with AcPseF displaying metal-dependent activity over a broad pH and temperature range. Upon binding to CMP-Pse5Ac7Ac, AcPseF undergoes dynamic movements akin to other CMP-ulosonic acid synthetases. The enzyme clearly discriminates Pse5Ac7Ac from other ulosonic acids, through active site interactions with side-chain functional groups and by positioning the molecule in a hydrophobic pocket. Finally, we show that AcPseF binds the CMP-Pse5Ac7Ac side chain in the lowest energy conformation, a trend that we observed in the structures of other enzymes of this class., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Translation-targeting toxic small alarmone synthetases (toxSAS) are effectors of bacterial toxin-antitoxin systems that pyrophosphorylate the 3'-CCA end of transfer RNA (tRNA) to prevent aminoacylation. toxSAS are implicated in antiphage immunity: Phage detection triggers the toxSAS activity to shut down viral production. We show that the toxSAS FaRel2 inspects the tRNA acceptor stem to specifically select tRNA Gly and tRNA Thr . The first, second, fourth, and fifth base pairs of the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRel SJ46 differ in tRNA specificity from FaRel2 and rationalize this through structural modeling: While the universal 3'-CCA end slots into a highly conserved CCA recognition groove, the acceptor stem recognition region is variable across toxSAS diversity. As phages use tRNA isoacceptors to overcome tRNA-targeting defenses, we hypothesize that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.

Auxins play a critical role in several plant developmental processes and their endogenous levels are regulated at multiple levels. The enzymes of the GRETCHEN HAGEN 3 (GH3) protein family catalyze the conjugation of amino acids to indoleacetic acid (IAA), the major endogenous auxin. The GH3 proteins are encoded by multiple redundant genes in plant genomes, making it difficult to perform functional genetic studies to understand their role in auxin homeostasis. To address these challenges, we used a chemical approach that exploits the reaction mechanism of GH3 proteins to identify small molecule inhibitors of their activity from a defined chemical library. The study evaluated receptor-ligand complexes based on their binding energy and classified them accordingly. Docking algorithms were used to correct any deviations, resulting in a list of the most important inhibitory compounds for selected GH3 enzymes based on a normalized sum of energy. The study presents atomic details of protein-ligand interactions and quantifies the effect of several of the identified small molecule inhibitors on auxin-mediated root growth processes in Arabidopsis thaliana. The direct effect of these compounds on endogenous auxin levels was measured using appropriate auxin sensors and endogenous hormone measurements. Our study has identified novel compounds of the flavonoid biosynthetic pathway that are effective inhibitors of GH3 enzyme-mediated IAA conjugation. These compounds play a versatile role in hormone-regulated plant development and have potential applications in both basic research and agriculture., (© 2024 Scandinavian Plant Physiology Society.)

G-Quadruplex/thioflavin (G4/THT) has become a very promising label-free fluorescent luminescent element for nucleic acid detection due to its good programmability and compatibility. However, the weak fluorescence efficiency of single-molecule G4/THT limits its potential applications. Here, we developed an entropy-driven catalytic (EDC) G4 (EDC-G4) cycle amplification technology as a universal label-free signal amplification and output system by properly programming classical EDC and G4 backbone sequences, preintegrated ligase chain reaction (LCR) for label-free sensitive detection of single nucleotide polymorphisms (SNPs). First, the positive strand LCR enabled specific transduction and preliminary signal amplification from single-base mutation information to single-strand information. Subsequently, the EDC-G4 cycle amplification reaction was activated, accompanied by the production of a large number of G4/THT luminophores to output fluorescent signals. The EDC-G4 system was proposed to address the weak fluorescence of G4/THT and obtain a label-free fluorescence signal amplification. The dual-signal amplification effect enabled the LCR-EDC-G4 detection system to accurately detect mutant target (MT) at concentrations as low as 22.39 fM and specifically identify 0.01% MT in a mixed detection pool. Moreover, the LCR-EDC-G4 system was further demonstrated for its potential application in real biological samples. Therefore, this study not only contributes ideas for the development of label-free fluorescent biosensing strategies but also provides a high-performance and practical SNP detection tool in parallel.

Naturally occurring lanthipeptides, peptides post-translationally modified by various enzymes, hold significant promise as antibiotics. Despite extensive biochemical and structural studies, the events preceding peptide modification remain poorly understood. Here, we identify a distinct subclass of lanthionine synthetase KC (LanKC) enzymes with distinct structural and functional characteristics. We show that PneKC, a member of this subclass, forms a dimer and possesses GTPase activity. Through three cryo-EM structures of PneKC, we illustrate different stages of peptide PneA binding, from initial recognition to full binding. Our structures show the kinase domain complexed with the PneA core peptide and GTPγS, a phosphate-bound lyase domain, and an unconventional cyclase domain. The leader peptide of PneA interact with a gate loop, transitioning from an extended to a helical conformation. We identify a dimerization hot spot and propose a "negative cooperativity" mechanism toggling the enzyme between tense and relaxed conformation. Additionally, we identify an important salt bridge in the cyclase domain, differing from those in in conventional cyclase domains. These residues are highly conserved in the LanKC subclass and are part of two signature motifs. These results unveil potential differences in lanthipeptide modification enzymes assembly and deepen our understanding of allostery in these multifunctional enzymes., (© 2024. The Author(s).)

Inteins are unique single-turnover enzymes that can excise themselves from the precursor protein without the aid of any external cofactors or energy. In most cases, inteins are covalently linked with the extein sequences and protein splicing happens spontaneously. In this study, a novel protein ligation system was developed based on two atypical split inteins without cross reaction, in which the large segments of one S1 and one S11 split intein fusion protein acted as a protein ligase, the small segments (only several amino acids long) was fused to the N-extein and C-extein, respectively. The splicing activity was demonstrated in E. coli and in vitro with different extein sequences, which showed ∼15% splicing efficiency in vitro. The protein trans-splicing in vitro was further optimized, and possible reaction explanations were explored. As a proof of concept, we expect this approach to expand the scope of trans-splicing-based protein engineering and provide new clues for intein based protein ligase., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay to identify more AEP-type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP-type peptide ligase activity in the crude extract of all tested bamboo leaves. From a popular bamboo species, Bambusa multiplex, we identified a full-length AEP-type peptide ligase candidate (BmAEP1) via transcriptomic sequencing. After its zymogen was overexpressed in Escherichia coli and self-activated in vitro, BmAEP1 displayed high peptide ligase activity, but with considerable hydrolytic activity. After site-directed mutagenesis of its ligase activity determinants, the mutant zymogen of [G238V]BmAEP1 was normally overexpressed in E. coli, but failed to activate itself. To resolve this problem, we developed a novel protease-assisted activation approach in which trypsin was used to cleave the mutant zymogen and was then conveniently removed via ion-exchange chromatography. After the noncovalently bound cap domain was dissociated from the catalytic core domain under acidic conditions, the recombinant [G238V]BmAEP1 displayed high peptide ligase activity with much lower hydrolytic activity and could efficiently catalyze inter-molecular protein ligation and intramolecular peptide cyclization. Thus, the engineered bamboo-derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis., (© 2024 Federation of European Biochemical Societies.)

GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetases conjugate amino acids to acyl acid hormones to either activate or inactivate the hormone molecule. The largest subgroup of GH3 proteins modify the growth-promoting hormone auxin (indole-3-acetic acid; IAA) with the second largest class activating the defense hormone jasmonic acid (JA). The two-step reaction mechanism of GH3 proteins provides a potential proofreading mechanism to ensure fidelity of hormone modification. Examining pyrophosphate release in the first-half reaction of Arabidopsis GH3 proteins that modify IAA (AtGH3.2/YDK2, AtGH3.5/WES1, AtGH3.17/VAS2), JA (AtGH3.11/JAR1), and other acyl acids (AtGH3.7, AtGH3.12/PBS3) indicates that acyl acid-AMP intermediates are hydrolyzed into acyl acid and AMP in the absence of the amino acid, a typical feature of pre-transfer editing mechanisms. Single-turnover kinetic analysis of AtGH3.2/YDK2 and AtGH3.5/WES1 shows that non-cognate acyl acid-adenylate intermediates are more rapidly hydrolyzed than the cognate IAA-adenylate. In contrast, AtGH3.11/JAR1 only adenylates JA, not IAA. While some of the auxin-conjugating GH3 proteins in Arabidopsis (i.e., AtGH3.5/WES1) accept multiple acyl acid substrates, others, like AtGH3.2/YDK2, are specific for IAA; however, both these proteins share similar active site residues. Biochemical analysis of chimeric variants of AtGH3.2/YDK2 and AtGH3.5/WES1 indicates that the C-terminal domain contributes to selection of cognate acyl acid substrates. These findings suggest that the hydrolysis of non-cognate acyl acid-adenylate intermediates, or proofreading, proceeds via a slowed structural switch that provides a checkpoint for fidelity before the full reaction proceeds., Competing Interests: Conflict of interest J. M. J. is an associate editor of this journal. The other author declares that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The biosynthesis of many bacterial siderophores employs a member of a family of ligases that have been defined as NRPS-independent siderophore (NIS) synthetases. These NIS synthetases use a molecule of ATP to produce an amide linkage between a carboxylate and an amine. Commonly used carboxylate substrates include citrate or α-ketoglutarate, or derivatives thereof, while the amines are often hydroxamate derivatives of lysine or ornithine, or their decarboxylated forms cadaverine and putrescine. Enzymes that employ three substrates to catalyze a reaction may proceed through alternate mechanisms. Some enzymes use sequential mechanisms in which all three substrates bind prior to any chemical steps. In such mechanisms, substrates can bind in a random, ordered, or mixed fashion. Alternately, other enzymes employ a ping-pong mechanism in which a chemical step occurs prior to the binding of all three substrates. Here we describe an enzyme assay that will distinguish among these different mechanisms for the NIS synthetase, using IucA, an enzyme involved in the production of aerobactin, as the model system., (Copyright © 2024. Published by Elsevier Inc.)

Two homologs of the diterpene synthase CotB2 from Streptomyces collinus (ScCotB2) and Streptomyces iakyrus (SiCotB2) were investigated for their products by in vitro incubations of the recombinant enzymes with geranylgeranyl pyrophosphate, followed by compound isolation and structure elucidation by NMR. ScCotB2 produced the new compound collinodiene, besides the canonical CotB2 product cyclooctat-9-en-7-ol, dolabella-3,7,18-triene and dolabella-3,7,12-triene, while SiCotB2 gave mainly cyclooctat-9-en-7-ol and only traces of dolabella-3,7,18-triene. The cyclisation mechanism towards the ScCotB2 products and their absolute configurations were investigated through isotopic labelling experiments., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Polymorphic toxins (PTs) are a broad family of toxins involved in interbacterial competition and pathogenesis. PTs are modular proteins that are comprised of a conserved N-terminal domain responsible for its transport, and a variable C-terminal domain bearing toxic activity. Although the mode of transport has yet to be elucidated, a new family of putative PTs containing an N-terminal MuF domain, resembling the Mu coliphage F protein, was identified in prophage genetic elements. The C-terminal toxin domains of these MuF PTs are predicted to bear nuclease, metallopeptidase, ADP-ribosyl transferase and RelA_SpoT activities. In this study, we characterized the MuF-RelA_SpoT toxin associated with the temperate phage of Streptococcus pneumoniae SPNA45. We show that the RelA_SpoT domain has (p)ppApp synthetase activity, which is bactericidal under our experimental conditions. We further determine that the two genes located downstream encode two immunity proteins, one binding to and inactivating the toxin and the other detoxifying the cell via a pppApp hydrolase activity. Finally, based on protein sequence alignments, we propose a signature for (p)ppApp synthetases that distinguishes them from (p)ppGpp synthetases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Sulfolipid-1 (SL-1) is a lipid that is abundantly found in the cell wall of Mycobacterium tuberculosis (Mtb). MtbFadD23 is crucial in the SL-1 synthesis pathway. Previously, 5'-O-[N-(11-phenoxyundecanoyl)sulfamoyl]adenosine (PhU-AMS) has been shown to be a general inhibitor of fatty-acid-adenylating enzymes (FadDs) in Mtb. However, the fatty acyl-AMP ligase (FAAL) class of FadDs, which includes MtbFadD23, appears to be functionally nonredundant in the production of multiple fatty acids. In this study, the ability of PhU-AMS to bind to MtbFadD23 was examined under in vitro conditions. The crystal structure of the MtbFadD23-PhU-AMS complex was determined at a resolution of 2.64 Å. Novel features were identified by structural analysis and comparison. Although PhU-AMS could bind to MtbFadD23, it did not inhibit the FAAL adenylation activity of MtbFadD23. However, PhU-AMS improved the main T m value in a differential scanning fluorimetry assay, and a structural comparison of MtbFadD23-PhU-AMS with FadD32 and PA1221 suggested that PhU-AMS blocks the loading of the acyl chain onto Pks2. This study sheds light on the structure-based design of specific inhibitors of MtbFadD23 and general inhibitors of FAALs.

Lanthipeptide synthetases are fascinating biosynthetic enzymes that install intramolecular thioether bridges into genetically encoded peptides, typically endowing the peptide with therapeutic properties. The factors that control the macrocyclic topology of lanthipeptides are numerous and remain difficult to predict and manipulate. The key challenge in this endeavor derives from the vast conformational space accessible to the disordered precursor lanthipeptide, which can be manipulated in subtle ways by interaction with the cognate synthetase. This review explores the unique strategies employed by each of the five phylogenetically divergent classes of lanthipeptide synthetase to manipulate and exploit the dynamic lanthipeptide conformational ensemble, collectively enabling these biosynthetic enzymes to guide peptide maturation along specific trajectories to products with distinct macrocyclic topology and biological activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Lanthipeptides are ribosomally-synthesized natural products from bacteria featuring stable thioether-crosslinks and various bioactivities. Herein, we report on a new clade of tricyclic class-IV lanthipeptides with curvocidin from Thermomonospora curvata as its first representative. We obtained crystal structures of the corresponding lanthipeptide synthetase CuvL that showed a circular arrangement of its kinase, lyase and cyclase domains, forming a central reaction chamber for the iterative substrate processing involving nine catalytic steps. The combination of experimental data and artificial intelligence-based structural models identified the N-terminal subdomain of the kinase domain as the primary site of substrate recruitment. The ribosomal precursor peptide of curvocidin employs an amphipathic α-helix in its leader region as an anchor to CuvL, while its substrate core shuttles within the central reaction chamber. Our study thus reveals general principles of domain organization and substrate recruitment of class-IV and class-III lanthipeptide synthetases., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

In recent years, some peptide ligases have been identified, such as bacterial sortases and certain plant asparaginyl or prolyl endopeptidases. Peptide ligases have wide applications in protein labelling and cyclic peptide synthesis. To characterize various known peptide ligases or identify new ones, we propose a general bioluminescent activity assay via the genetic fusion of a recognition motif of peptide ligase(s) to the C-terminus of an inactive large NanoLuc fragment (LgBiT) and the chemical introduction of a nucleophilic motif preferred by the peptide ligase(s) to the N-terminus of the low-affinity SmBiT complementation tag. After the inactive ligation version LgBiT protein was ligated with the low-affinity ligation version SmBiT tag by the expected peptide ligase(s), its luciferase activity would be restored and could be quantified sensitively according to the measured bioluminescence. In the present study, we first validated the bioluminescent activity assay using bacterial sortase A and plant-derived butelase-1. Subsequently, we screened novel peptide ligases from crude extracts of selected plants using two LgBiT-SmBiT ligation pairs. Among 80 common higher plants, we identified that five of them likely express asparaginyl endopeptidase-type peptide ligase and four of them likely express prolyl endopeptidase-type peptide ligase, suggesting that peptide ligases are not so rare in higher plants and more of them await discovery. The present bioluminescent activity assay is ultrasensitive, convenient for use, and resistant to protease interference, and thus would have wide applications for characterizing known peptide ligases or screening new ones from various sources in future studies., (© 2022 Federation of European Biochemical Societies.)

GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetases catalyze the ATP-dependent conjugation of phytohormones with amino acids. Traditionally, GH3 proteins are associated with synthesis of the bioactive jasmonate hormone (+)-7- iso -jasmonoyl-l-isoleucine (JA-Ile) and conjugation of indole-3-acetic acid (IAA) with amino acids that tag the hormone for degradation and/or storage. Modifications of JA and IAA by GH3 acyl acid amido synthetases help maintain phytohormones homeostasis. Recent studies broaden the roles of GH3 proteins to include the regulation of JA biosynthesis; the modification of other auxins (i.e., phenylacetic acid and indole-3-butyric acid); the conjugation of auxinic herbicides, such as 4-dichlorophenoxyacetic acid, 4-(2,4-dichlorophenoxy)butyric acid, and dicamba; and the missing step in the isochorismate pathway for the biosynthesis of salicylic acid. The GH3 protein family joins the growing number of versatile enzyme families that blur the line between primary and specialized metabolism for an increasing range of biology functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Class II lanthipeptide synthetases (LanMs) are relatively promiscuous to core peptide variations. Previous studies have shown that different LanMs catalyze identical reactions on the same core sequence fused to their respective cognate leaders. We characterized a new LanM enzyme from Microcystis aeruginosa NIES-88, MalM, and demonstrated that MalM and ProcM exhibited disparate dehydration and cyclization patterns on identical core peptides. Our study provided new insights into the regioselectivity of LanMs and showcased an appropriate strategy for lanthipeptide structural diversity engineering.

The synthesis of amides through acid and amine coupling is one of the most commonly used reactions in medicinal chemistry, yet still requires atom-inefficient coupling reagents. There is a current demand to develop greener, biocatalytic approaches to amide bond formation. The nitrile synthetase (NS) enzymes are a small family of ATP-dependent enzymes which catalyse the transformation of a carboxylic acid into the corresponding nitrile via an amide intermediate. The Bacillus subtilis QueC (BsQueC) is an NS involved in the synthesis of 7-cyano-7-deazaguanine (CDG) natural products. Through sequence homology and structural analysis of BsQueC we identified three highly conserved residues, which could potentially play important roles in NS substrate binding and catalysis. Rational engineering led to the creation of a NS K163A/R204A biocatalyst that converts the CDG acid into the primary amide, but does not proceed to the nitrile. This study suggests that NSs could be further developed for coupling agent-free, amide-forming biocatalysts., (© 2021 Wiley-VCH GmbH.)

UDP-Xyl, a nucleotide sugar involved in the biosynthesis of various glycoconjugates, is difficult to obtain and quite expensive. Biocatalysis using a one-pot multi-enzyme cascade is one of the most valuable biotransformation processes widely used in the industry. Herein, two enzymes, UDP-glucose (UDP-Glc) dehydrogenase (CGIUGD) and UDP-Xyl synthase (CGIUXS) from the Pacific oyster Crassostrea gigas, which are coupled together for the biotransformation of UDP-Xyl, were characterized. The optimum pH was determined to be pH 9.0 for CGIUGD and pH 7.5 for CGIUXS. Both enzymes showed the highest activity at 37 °C. Neither enzyme is metal ion-dependent. On this basis, a single factor and orthogonal test were applied to optimize the condition of biotransformation of UDP-Xyl from UDP-Glc. Orthogonal design L9 (3 3 ) was conducted to optimize processing variables of enzyme amount, pH, and temperature. The conversion of UDP-Xyl was selected as an analysis indicator. Optimum variables were the ratio of CGIUGD to CGIUXS of 2:5, enzymatic pH of 8.0, and temperature of 37 °C, which is confirmed by three repeated validation experiments. The UDP-Xyl conversion was 69.921% in a 1 mL reaction mixture by optimized condition for 1 h. This is the first report for the biosynthesis of UDP-Xyl from oyster enzymes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS's catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel-Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Peptidyl Asx-specific ligases (PALs) effect peptide ligation by catalyzing transpeptidation reactions at Asn/Asp-peptide bonds. Owing to their high efficiency and mild aqueous reaction conditions, these ligases have emerged as powerful biotechnological tools for protein manipulation in recent years. PALs are enzymes of the asparaginyl endopeptidase (AEP) superfamily but have predominant transpeptidase activity as opposed to typical AEPs which are predominantly hydrolases. Butelase-1 and VyPAL2, two PALs discovered by our teams, have been used successfully in a wide range of applications, including macrocyclization of synthetic peptides and recombinant proteins, protein N- or C-terminal modification, and cell-surface labeling. As shown in numerous reports, PAL-mediated ligation is highly efficient at Asn junctions. Although considerably less efficient, Asp-specific ligation has also been shown to be practically useful under suitable conditions. Herein, we describe the methods of using VyPAL2 for protein macrocyclization and labeling at an Asp residue as well as for protein dual labeling through orthogonal Asp- and Asn-directed ligations. We also describe a method for cell-surface protein modification using butelase-1, demonstrating its advantageous features over previous methods., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The last two decades have seen an increasing demand for new protein-modification methods from the biotech industry and biomedical research communities. Owing to their mild aqueous reaction conditions, enzymatic methods based on the use of peptide ligases are particularly desirable. In this regard, the recently discovered peptidyl Asx-specific ligases (PALs) have emerged as powerful biotechnological tools in recent years. However, as a new class of peptide ligases, their scope and application remain underexplored. Herein, we report the use of a new PAL, VyPAL2, for a diverse range of protein modifications. We successfully showed that VyPAL2 was an efficient biocatalyst for protein labelling, inter-protein ligation, and protein cyclization. The labelled or cyclized protein ligands remained functionally active in binding to their target receptors. We also demonstrated on-cell labelling of protein ligands pre-bound to cellular receptors and cell-surface engineering via modifying a covalently anchored peptide substrate pre-installed on cell-surface glycans. Together, these examples firmly establish Asx-specific ligases, such as VyPAL2, as the biocatalysts of the future for site-specific protein modification, with a myriad of applications in basic research and drug discovery.

Graspetides are a class of ribosomally synthesized and post-translationally modified peptide natural products featuring ATP-grasp ligase-dependent formation of macrolactones/macrolactams. These modifications arise from serine, threonine, or lysine donor residues linked to aspartate or glutamate acceptor residues. Characterized graspetides include serine protease inhibitors such as the microviridins and plesiocin. Here, we report an update to Rapid ORF Description and Evaluation Online (RODEO) for the automated detection of graspetides, which identified 3,923 high-confidence graspetide biosynthetic gene clusters. Sequence and co-occurrence analyses doubled the number of graspetide groups from 12 to 24, defined based on core consensus sequence and putative secondary modification. Bioinformatic analyses of the ATP-grasp ligase superfamily suggest that extant graspetide synthetases diverged once from an ancestral ATP-grasp ligase and later evolved to introduce a variety of ring connectivities. Furthermore, we characterized thatisin and iso -thatisin, two graspetides related by conformational stereoisomerism from Lysobacter antibioticus . Derived from a newly identified graspetide group, thatisin and iso -thatisin feature two interlocking macrolactones with identical ring connectivity, as determined by a combination of tandem mass spectrometry (MS/MS), methanolytic, and mutational analyses. NMR spectroscopy of thatisin revealed a cis conformation for a key proline residue, while molecular dynamics simulations, solvent-accessible surface area calculations, and partial methanolytic analysis coupled with MS/MS support a trans conformation for iso -thatisin at the same position. Overall, this work provides a comprehensive overview of the graspetide landscape, and the improved RODEO algorithm will accelerate future graspetide discoveries by enabling open-access analysis of existing and emerging genomes.

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning–based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.

With the redefinition of polyketide synthase (PKS) modules, a new appreciation of their most downstream domain, the ketosynthase (KS), is emerging. In addition to performing its well-established role of generating a carbon-carbon bond between an acyl-CoA building block and a growing polyketide, it may gatekeep against incompletely processed intermediates. Here, we investigate 739 KSs from 92 primarily actinomycete, cis -acyltransferase assembly lines. When KSs were separated into 16 families based on the chemistries at the α- and β-carbons of their polyketide substrates, a comparison of 32 substrate tunnel residues revealed unique sequence fingerprints. Surprisingly, additional fingerprints were detected when the chemistry at the γ-carbon was considered. Representative KSs were modeled bound to their natural polyketide substrates to better understand observed patterns, such as the substitution of a tryptophan by a smaller residue to accommodate an l-α-methyl group or the substitution of four smaller residues by larger ones to make better contact with a primer unit or diketide. Mutagenesis of a conserved glutamine in a KS within a model triketide synthase indicates that the substrate tunnel is sensitive to alteration and that engineering this KS to accept unnatural substrates may require several mutations.

Graspetides, also known as ω-ester-containing peptides (OEPs), are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) bearing side chain-to-side chain macrolactone or macrolactam linkages. Here, we present the molecular details of precursor peptide recognition by the macrocyclase enzyme PsnB in the biosynthesis of plesiocin, a group 2 graspetide. Biochemical analysis revealed that, in contrast to other RiPPs, the core region of the plesiocin precursor peptide noticeably enhanced the enzyme-precursor interaction via the conserved glutamate residues. We obtained four crystal structures of symmetric or asymmetric PsnB dimers, including those with a bound core peptide and a nucleotide, and suggest that the highly conserved Arg213 at the enzyme active site specifically recognizes a ring-forming acidic residue before phosphorylation. Collectively, this study provides insights into the mechanism underlying substrate recognition in graspetide biosynthesis and lays a foundation for engineering new variants., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Bioconjugation is an important chemical biology research focus, especially in the development of methods to produce pharmaceutical bioconjugates and antibody-drug conjugates (ADCs). In this report, an enzyme-catalyzed conjugation method combined with a chemical reaction was used to modify a native antibody under mild reaction conditions. Our investigation revealed that lipoic-acid ligase (LplA) modifies native IgG1 with biased site-specificity. An intact mass analysis revealed that 98.3% of IgG1 was modified by LplA and possessed at least one molecule of octanocic acid. The average number of modifications per antibody was calculated to be 4.6. Peptide mapping analysis revealed that the modified residues were K225, K249 and K363 in the Fc region, and K30, K76 and K136 in the heavy chain and K39/K42, K169, K188 and K190 in the light chain of the Fab region. Careful evaluation including solvent exposed amino acid analysis suggested that these conjugate sites were not only solvent exposed but also biased by the site-specificity of LplA. Furthermore, antibody fragment conjugation may be able to take advantage of this enzymatic approach. This feasibility study serves as a demonstration for preparing enzymatically modified antibodies with conjugation site analysis., (Copyright © 2021. Published by Elsevier Ltd.)

A method based on mRNA-templated ligation of splice-junction anchored DNA probes followed by PCR amplification of the ligated product has been developed for multiplexed detection of mRNA splice variants with high sensitivity and specificity. The proposed assay can detect as low as 10 aM mRNA splicing variants and has been successfully applied to detect real samples.

Fatty acyl-AMP ligases (FAALs) channelize fatty acids towards biosynthesis of virulent lipids in mycobacteria and other pharmaceutically or ecologically important polyketides and lipopeptides in other microbes. They do so by bypassing the ubiquitous coenzyme A-dependent activation and rely on the acyl carrier protein-tethered 4'-phosphopantetheine ( holo -ACP). The molecular basis of how FAALs strictly reject chemically identical and abundant acceptors like coenzyme A (CoA) and accept holo -ACP unlike other members of the ANL superfamily remains elusive. We show that FAALs have plugged the promiscuous canonical CoA-binding pockets and utilize highly selective alternative binding sites. These alternative pockets can distinguish adenosine 3',5'-bisphosphate-containing CoA from holo -ACP and thus FAALs can distinguish between CoA and holo -ACP. These exclusive features helped identify the omnipresence of FAAL-like proteins and their emergence in plants, fungi, and animals with unconventional domain organizations. The universal distribution of FAALs suggests that they are parallelly evolved with FACLs for ensuring a CoA-independent activation and redirection of fatty acids towards lipidic metabolites., Competing Interests: GP, SM, SS, KP, SP, ND, SN, MM, BP, RG None, PK none, RS Reviewing editor, eLife, (© 2021, Patil et al.)

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glycosynthases are glycoside hydrolase mutants that can synthesize oligosaccharides or glycosides from an inverted donor without hydrolysis of the products. Although glycosynthases have been characterized from a variety of glycoside hydrolase (GH) families, family GH116 glycosynthases have yet to be reported. We produced the Thermoanaerobacterium xylanolyticum TxGH116 nucleophile mutants E441D, E441G, E441Q and E441S and compared their glycosynthase activities to the previously generated E441A mutant. The TxGH116 E441G and E441S mutants exhibited highest glycosynthase activity to transfer glucose from α-fluoroglucoside (α-GlcF) to cellobiose acceptor, while E441D had low but significant activity as well. The E441G, E441S and E441A variants showed broad specificity for α-glycosyl fluoride donors and p-nitrophenyl glycoside acceptors. The structure of the TxGH116 E441A mutant with α-GlcF provided the donor substrate complex, while soaking of the TxGH116 E441G mutant with α-GlcF resulted in cellooligosaccharides extending from the +1 subsite out of the active site, with glycerol in the -1 subsite. Soaking of E441A or E441G with cellobiose or cellotriose gave similar acceptor substrate complexes with the nonreducing glucosyl residue in the +1 subsite. Combining structures with the ligands from the TxGH116 E441A with α-GlcF crystals with that of E441A or E441G with cellobiose provides a plausible structure of the catalytic ternary complex, which places the nonreducing glucosyl residue O4 2.5 Å from the anomeric carbon of α-GlcF, thereby explaining its apparent preference for production of β-1,4-linked oligosaccharides. This functional and structural characterization provides the background for development of GH116 glycosynthases for synthesis of oligosaccharides and glycosides of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Natural [4 + 2]-cyclases catalyze concerted cycloaddition during biosynthesis of over 400 natural products reported. Microbial [4 + 2]-cyclases are structurally diverse with a broad range of substrates. Thus far, about 52 putative microbial [4 + 2]-cyclases of 13 different types have been characterized, with over 20 crystal structures. However, how these cyclases have evolved during natural product biosynthesis remains elusive. Structural and phylogenetic analyses suggest that these different types of [4 + 2]-cyclases might have diverse evolutionary origins, such as reductases, dehydratases, methyltransferases, oxidases, etc. Divergent evolution of enzyme function might have occurred in these different families. Understanding the independent evolutionary history of these cyclases would provide new insights into their catalysis mechanisms and the biocatalyst design., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Aeromonas hydrophila is a gram-negative bacterium and the major causative agent of the fish disease motile aeromonad septicemia (MAS). It uses N-acyl-homoserine lactone (AHL) quorum sensing signals to coordinate biofilm formation, motility, and virulence gene expression. The AHL signaling pathway is therefore considered to be a therapeutic target against pathogenic A. hydrophila infection. In A. hydrophila, AHL autoinducers biosynthesis are specifically catalyzed by an ACP-dependent AHL synthase AhyI using the precursors SAM and acyl-ACP. Our previously reported AhyI was heterologously expressed in E. coli, which showed the production characteristics of medium-long chain AHLs. This contradicted the prevailing understanding that AhyI was only a short-chain C 4 /C 6 -HSL synthase., Results: In this study, six linear acyl-ACP proteins with C-terminal his-tags were synthesized in Vibrio harveyi AasS using fatty acids and E. coli produced active holo-ACP proteins, and in vitro biosynthetic assays of six AHL molecules and kinetic studies of recombinant AhyI with a panel of four linear acyl-ACPs were performed. UPLC-MS/MS analyses indicated that AhyI can synthesize short-, medium- and long-chain AHLs from SAM and corresponding linear acyl-ACP substrates. Kinetic parameters measured using a DCPIP colorimetric assay, showed that there was a notable decrease in catalytic efficiency with acyl-chain lengths above C6, and hyperbolic or sigmoidal responses in rate curves were observed for varying acyl-donor substrates. Primary sequence alignment of the six representative AHL synthases offers insights into the structural basis for their specific acyl substrate preference. To further understand the acyl chain length preference of AhyI for linear acyl-ACP, we performed a structural comparison of three ACP-dependent LuxI homologs (TofI, BmaI1 and AhyI) and identified three key hydrophobic residues (I67, F125 and L157) which confer AhyI to selectively recognize native C 4 /C 6 -ACP substrates. These predictions were further supported by a computational Ala mutation assay., Conclusions: In this study, we have redefined AhyI as a multiple short- to long-chain AHL synthase which uses C 4 /C 6 -ACP as native acyl substrates and longer acyl-ACPs (C8 ~ C14) as non-native ones. We also theorized that the key residues in AhyI would likely drive acyl-ACP selective recognition.

Among the ribosomally synthesized and post-translationally modified peptide (RiPP) natural products, "graspetides" (formerly known as microviridins) contain macrocyclic esters and amides that are formed by ATP-grasp ligase tailoring enzymes using the side chains of Asp/Glu as acceptors and Thr/Ser/Lys as donors. Graspetides exhibit diverse patterns of macrocylization and connectivities exemplified by microviridins, that have a caged tricyclic core, and thuringin and plesiocin that feature a "hairpin topology" with cross-strand ω-ester bonds. Here, we characterize chryseoviridin, a new type of multicore RiPP encoded by Chryseobacterium gregarium DS19109 (Phylum Bacteroidetes) and solve a 2.44 Å resolution crystal structure of a quaternary complex consisting of the ATP-grasp ligase CdnC bound to ADP, a conserved leader peptide and a peptide substrate. HRMS/MS analyses show that chryseoviridin contains four consecutive five- or six-residue macrocycles ending with a microviridin-like core. The crystal structure captures respective subunits of the CdnC homodimer in the apo or substrate-bound state revealing a large conformational change in the B-domain upon substrate binding. A docked model of ATP places the γ-phosphate group within 2.8 Å of the Asp acceptor residue. The orientation of the bound substrate is consistent with a model in which macrocyclization occurs in the N- to C-terminal direction for core peptides containing multiple Thr/Ser-to-Asp macrocycles. Using systematically varied sequences, we validate this model and identify two- or three-amino acid templating elements that flank the macrolactone and are required for enzyme activity in vitro. This work reveals the structural basis for ω-ester bond formation in RiPP biosynthesis.

DNA curvature is the result of a combination of both intrinsic features of the double helix and external distortions introduced by the environment and the binding of proteins or drugs. The propensity of certain double-stranded DNA (dsDNA) sequences to bend is essential in crucial biological processes, such as replication and transcription, in which proteins are known to either recognize noncanonical DNA conformations or promote their formation upon DNA binding. Trabectedin (Yondelis®) is a clinically used antitumor drug which, following covalent bond formation with the 2-amino group of guanine, induces DNA curvature and enhances the circularization ratio, upon DNA ligation, of several dsDNA constructs but not others. By means of unrestrained molecular dynamics simulations using explicitly solvated all-atom models, we rationalize these experimental findings in structural terms and shed light on the crucial, albeit possibly underappreciated, role played by T4 DNA ligase in stabilizing a bent DNA conformation prior to cyclization. Taken together, our results expand our current understanding on how DNA shape modification by trabectedin may affect both the sequence-specific recognition by transcription factors to promoter sites and RNA polymerase II binding.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. These genetically encoded peptides are biosynthesized by multifunctional enzymes (lanthipeptide synthetases) that possess relaxed substrate specificity and catalyze iterative rounds of post-translational modification. Recent evidence has suggested that some lanthipeptide synthetases are structurally dynamic enzymes that are allosterically activated by precursor peptide binding and that conformational sampling of the enzyme-peptide complex may play an important role in defining the efficiency and sequence of biosynthetic events. These "biophysical" processes, while critical for defining the activity and function of the synthetase, remain very challenging to study with existing methodologies. Herein, we show that native mass spectrometry coupled to ion mobility (native IM-MS) provides a powerful and sensitive means for investigating the conformational landscapes and intermolecular interactions of lanthipeptide synthetases. Namely, we demonstrate that the class II lanthipeptide synthetase (HalM2) and its noncovalent complex with the cognate HalA2 precursor peptide can be delivered into the gas phase in a manner that preserves native structures and intermolecular enzyme-peptide contacts. Moreover, gas phase ion mobility studies of the natively folded ions demonstrate that peptide binding and mutations to dynamic structural elements of HalM2 alter the conformational landscape of the enzyme. Cumulatively, these data support previous claims that lanthipeptide synthetases are structurally dynamic enzymes that undergo functionally relevant conformational changes in response to precursor peptide binding. This work establishes native IM-MS as a versatile approach for characterizing intermolecular interactions and for unraveling the relationships between protein structure and biochemical function in RiPP biosynthetic systems.

Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl-L-isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways 1-4 . Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown 1,2 . Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and L-Ile in plants 5 . This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions-Jar1 producing a compound essential for plant development 4,5 , and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications.

Peptidoglycan comprises repeating units of N-acetylmuramic acid, N-acetylglucosamine and short cross-linking peptides. After the conversion of UDP-N-acetylglucosamine (UNAG) to UDP-N-acetylmuramic acid (UNAM) by the MurA and MurB enzymes, an amino acid is added to UNAM by UDP-N-acetylmuramic acid L-alanine ligase (MurC). As peptidoglycan is an essential component of the bacterial cell wall, the enzymes involved in its biosynthesis represent promising targets for the development of novel antibacterial drugs. Here, the crystal structure of Mycobacterium bovis MurC (MbMurC) is reported, which exhibits a three-domain architecture for the binding of UNAM, ATP and an amino acid as substrates, with a nickel ion at the domain interface. The ATP-binding loop adopts a conformation that is not seen in other MurCs. In the UNAG-bound structure of MbMurC, the substrate mimic interacts with the UDP-binding domain of MbMurC, which does not invoke rearrangement of the three domains. Interestingly, the glycine-rich loop of the UDP-binding domain of MbMurC interacts through hydrogen bonds with the glucose moiety of the ligand, but not with the pyrophosphate moiety. These findings suggest that UNAG analogs might serve as potential candidates for neutralizing the catalytic activity of bacterial MurC.

The stringent response, regulated by the bifunctional (p)ppGpp synthetase/hydrolase Rel in mycobacteria, is critical for long-term survival of the drug-tolerant dormant state of Mycobacterium tuberculosis. During amino acid starvation, MtRel senses a drop in amino acid concentration and synthesizes the messengers pppGpp and ppGpp, collectively called (p)ppGpp. Here, we investigate the role of the regulatory 'Aspartokinase, Chorismate mutase and TyrA' (ACT) domain in MtRel. Using NMR spectroscopy approaches, we report the high-resolution structure of dimeric MtRel ACT which selectively binds to valine out of all other branched-chain amino acids tested. A set of MtRel ACT mutants were generated to identify the residues required for maintaining the head-to-tail dimer. Through NMR titrations, we determined the crucial residues for binding of valine and show structural rearrangement of the MtRel ACT dimer in the presence of valine. This study suggests the direct involvement of amino acids in (p)ppGpp accumulation mediated by MtRel independent to interactions with stalled ribosomes. Database Structural data are available in the PDB database under the accession number 6LXG., (© 2020 Federation of European Biochemical Societies.)

The polyketide natural product reveromycin A (RM-A) exhibits antifungal, anticancer, anti-bone metastasis, anti-periodontitis and anti-osteoporosis activities by selectively inhibiting eukaryotic cytoplasmic isoleucyl-tRNA synthetase (IleRS). Herein, a co-crystal structure suggests that the RM-A molecule occupies the substrate tRNA Ile binding site of Saccharomyces cerevisiae IleRS (ScIleRS), by partially mimicking the binding of tRNA Ile . RM-A binding is facilitated by the copurified intermediate product isoleucyl-adenylate (Ile-AMP). The binding assays confirm that RM-A competes with tRNA Ile while binding synergistically with L-isoleucine or intermediate analogue Ile-AMS to the aminoacylation pocket of ScIleRS. This study highlights that the vast tRNA binding site of the Rossmann-fold catalytic domain of class I aminoacyl-tRNA synthetases could be targeted by a small molecule. This finding will inform future rational drug design.

The understanding of how proteins evolve to perform novel functions has long been sought by biologists. In this regard, two homologous bacterial enzymes, PafA and Dop, pose an insightful case study, as both rely on similar mechanistic properties, yet catalyze different reactions. PafA conjugates a small protein tag to target proteins, whereas Dop removes the tag by hydrolysis. Given that both enzymes present a similar fold and high sequence similarity, we sought to identify the differences in the amino acid sequence and folding responsible for each distinct activity. We tackled this question using analysis of sequence-function relationships, and identified a set of uniquely conserved residues in each enzyme. Reciprocal mutagenesis of the hydrolase, Dop, completely abolished the native activity, at the same time yielding a catalytically active ligase. Based on the available Dop and PafA crystal structures, this change of activity required a conformational change of a critical loop at the vicinity of the active site. We identified the conserved positions essential for stabilization of the alternative loop conformation, and tracked alternative mutational pathways that lead to a change in activity. Remarkably, all these pathways were combined in the evolution of PafA and Dop, despite their redundant effect on activity. Overall, we identified the residues and structural elements in PafA and Dop responsible for their activity differences. This analysis delineated, in molecular terms, the changes required for the emergence of a new catalytic function from a preexisting one., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Class II lanthipeptide synthetases (LanM enzymes) catalyze the multistep post-translational modification of genetically encoded precursor peptides into macrocyclic (often antimicrobial) lanthipeptides. The reaction sequence involves dehydration of serine/threonine residues, followed by intramolecular addition of cysteine thiols onto the nascent dehydration sites to construct thioether bridges. LanMs utilize two separate active sites in an iterative yet highly coordinated manner to maintain a remarkable level of regio- and stereochemical control over the multistep maturation. The mechanisms underlying this biosynthetic fidelity remain enigmatic. We recently demonstrated that proper function of the haloduracin β synthetase (HalM2) requires dynamic structural elements scattered across the surface of the enzyme. Here, we perform kinetic simulations, structural analysis of reaction intermediates, hydrogen-deuterium exchange mass spectrometry studies, and molecular dynamics simulations to investigate the contributions of these dynamic HalM2 structural elements to biosynthetic efficiency and fidelity. Our studies demonstrate that a large, conserved loop (HalM2 residues P349-P405) plays essential roles in defining the precursor peptide binding site, facilitating efficient peptide dehydration, and guiding the order of thioether ring formation. Moreover, mutations near the interface of the HalM2 dehydratase and cyclase domains perturb cyclization fidelity and result in aberrant thioether topologies that cannot be corrected by the wild type enzyme, suggesting an element of kinetic control in the normal cyclization sequence. Overall, this work provides the most comprehensive correlation of the structural and functional properties of a LanM enzyme reported to date and should inform mechanistic studies of the biosynthesis of other ribosomally synthesized and post-translationally modified peptide natural products.

Considered a key taxon in soil and marine microbial communities, myxobacteria exist as coordinated swarms that utilize a combination of lytic enzymes and specialized metabolites to facilitate predation of microbes. This capacity to produce specialized metabolites and the associated abundance of biosynthetic pathways contained within their genomes have motivated continued drug discovery efforts from myxobacteria. Of all myxobacterial biosynthetic gene clusters deposited in the antiSMASH database, only one putative acylhomoserine lactone (AHL) synthase, agpI, was observed, in genome data from Archangium gephyra. Without an AHL receptor also apparent in the genome of A. gephyra, we sought to determine if AgpI was an uncommon example of an orphaned AHL synthase. Herein we report the bioinformatic assessment of AgpI and discovery of a second AHL synthase from Vitiosangium sp. During axenic cultivation conditions, no detectible AHL metabolites were observed in A. gephyra extracts. However, heterologous expression of each synthase in Escherichia coli provided detectible quantities of 3 AHL signals including 2 known AHLs, C8-AHL and C9-AHL. These results suggest that A. gephyra AHL production is dormant during axenic cultivation. The functional, orphaned AHL synthase, AgpI, is unique to A. gephyra, and its utility to the predatory myxobacterium remains unknown.

Resistance to antibiotics is a serious concern to treat infectious diseases and also, for food preservation. Existing antibiotics generally inhibit enzymes participating in key bacterial processes, such as formation of cell wall, replication, transcription and translation. However, bacteria have rapidly evolved new mechanisms to combat these antibiotics and it hence becomes indispensable to identify newer targets and identify/design inhibitors against them. Another concern is that most antibiotics are broad spectrum; they largely bind and inhibit the active site of the target enzyme. Rel proteins, which synthesize (and hydrolyze) (p)ppGpp in response to a variety of stress encountered by bacteria, is a profitable target owing to its distinct absence in humans and an intricate regulation of the catalytic activities. Inactivation of (p)ppGpp synthesis by Rel, disables bacterial survival in Mycobacterium tuberculosis and Staphylococcus aureus, while inactivating the hydrolysis activity was lethal. The poor MIC values of the currently known Rel inhibitors present a distinct opportunity to develop better inhibitors and warrants a detailed structural characterization and understanding of the complex regulation in Rel proteins. It will open new avenues for the design of effective, species-specific inhibitors. In an attempt to identify unique sites for inhibitor design using structure-based approaches, we initiate a study of Rel homologues from four different pathogenic bacteria, in order to compare their attributes with well characterized Rel homologues. Here, we present cloning, over-expression, purification and preliminary characterization of these four homologues; and suggest similarities and differences that can be exploited for inhibitor design., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Gretchen Hagen 3 (GH3) family of acyl acid amido synthetases regulate the levels and activities of plant hormones containing carboxyl groups, thereby modulating diverse physiological responses. While structure-function relationships have been elucidated for dicotyledonous GH3s, the catalytic mechanism of monocotyledonous GH3 remains elusive. Rice (Oryza sativa) is a representative monocot, and its yield is controlled by the natural growth hormone IAA (indole-3-acetic acid). OsGH3-8 is a model GH3 enzyme that conjugates excess IAA to amino acids in an ATP-dependent manner, ensuring auxin homeostasis and regulating disease resistance, growth and development. Here, we report the crystal structure of OsGH3-8 protein in complex with AMP to uncover the molecular and structural basis for the activity of monocotyledonous GH3-8. Structural and sequence comparisons with other GH3 proteins reveal that the AMP/ATP binding sites are highly conserved. Molecular docking studies with IAA, the GH3-inhibitor Adenosine-5'-[2-(1H-indol-3-yl)ethyl]phosphate (AIEP), and Aspartate provide important information for substrate binding and selectivity of OsGH3-8. Moreover, the observation that AIEP nearly occupies the entire binding site for AMP, IAA and amino acid, offers a ready explanation for the inhibitory effect of AIEP. Taken together, the present study provides vital insights into the molecular mechanisms of monocot GH3 function, and will help to shape the future designs of effective inhibitors., Competing Interests: Declaration of competing interest No conflict of interest exits in the submission of this manuscript and the manuscript is approved by all authors for publication., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In the present work, we investigated the minimal inhibitory concentration (MIC) against fungal strains (Fonsecaea pedrosoi, Microsporum canis, Candida albicans, Cryptococcus neoformans), and cytotoxicity to normal cell lines for modified red angico gum (AG) with eterifying agent N-chloride (3-chloro-2-hydroxypropyl) trimethylammonium (CHPTAC). Quaternized ammonium groups were linked to AG backbone using N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride. The chemical features of the quaternized gum derivatives (QAG) were analyzed by: FTIR, elemental analysis, Zeta potential and gel permeation chromatography. The angico quaternizated gum presented a degree of substitution (DS) of 0.22 and Zeta potential of +36.43. For the antifungal test, it was observed that unmodified gum did not inhibit fungal growth. While, QAG inhibited the growth of most fungi used in this study. By AFM technique QAG interacted with the fungal surface, altering wall roughness significantly. The probable affinity of fragments of the QAG structure for the fungal enzyme 5I33 (Adenylosuccinate synthetase) has been shown by molecular docking. Low cytotoxicity was observed for polymers (unmodified gum and QAG). The results demonstrate that the quaternized polymer of AG presented in this study is a quite promising biomaterial for biotechnological applications., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2018. Published by Elsevier B.V.)

Objective: To enhance adipic acid production, a computer-aided approach was employed to engineer the adipyl-CoA synthetase from Thermobifida fusca by combining sequence analysis, protein structure modeling, in silico site-directed mutagenesis, and molecular dynamics simulation., Results: Two single mutants of T. fusca adipyl-CoA synthetase, E210βN and E210βQ, achieved a specific enzyme activity of 1.95 and 1.84 U/mg, respectively, which compared favorably with the 1.48 U/mg for the wild-type. The laboratory-level fermentation experiments showed that E210βN and E210βQ achieved a maximum adipic acid titer of 0.32 and 0.3 g/L. In contrast, the wild-type enzyme yielded a titer of 0.15 g/L under the same conditions. Molecular dynamics (MD) simulations revealed that the mutants (E210βN and E210βQ) could accelerate the dephosphorylation process in catalysis and enhance enzyme activity., Conclusions: The combined computational-experimental approach provides an effective strategy for enhancing enzymatic characteristics, and the mutants may find a useful application for producing adipic acid.

Protein lipoylation is essential for the function of many key enzymes but barely studied kinetically. Here, the two-step reaction cascade of H protein lipoylation catalyzed by the multifunctional enzyme lipoate-protein ligase A (LplA) was quantitatively and differentially studied. We discovered new phenomena and unusual kinetics of the cascade: (a) the speed of the first reaction is faster than the second one by two orders of magnitude, leading to high accumulation of the intermediate lipoyl-AMP (Lip-AMP); (b) Lip-AMP is hydrolyzed, but only significantly at the presence of H protein and in competition with the lipoylation; (c) both the lipoylation of H protein and its hydrolysis is enhanced by the apo and lipoylated forms of H protein and a mutant without the lipoylation site. A conceptual mechanistic model is proposed to explain these experimental observations in which conformational change of LplA upon interaction with H protein and competitive nucleophilic attacks play key roles., (© 2020 Wiley Periodicals LLC.)

Histidine decarboxylase (HDC; EC 4.1.1.22), an enzyme that catalyzes histamine synthesis with high substrate specificity, is a member of the group II pyridoxal 5'-phosphate (PLP) -dependent decarboxylase family. Tyrosine is a conserved residue among group II PLP-dependent decarboxylases. Human HDC has a Y334 located on a catalytically important loop at the active site. In this study, we demonstrated that a HDC Y334F mutant is capable of catalyzing the decarboxylation-dependent oxidative deamination of histidine to yield imidazole acetaldehyde. Replacement of the active-site Tyr with Phe in group II PLP-dependent decarboxylases, including mammalian aromatic amino acid decarboxylase, plant tyrosine/DOPA decarboxylase, and plant tryptophan decarboxylase, is expected to result in the same functional change, given that a Y-to-F substitution at the corresponding residue (number 260) in the HDC of Morganella morganii, another group II PLP-dependent decarboxylase, yielded the same effect. Thus, it was suggested that the loss of the OH moiety from the active-site Tyr residue of decarboxylase uniquely converts the enzyme to an aldehyde synthase., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Our current understanding of the chemistry of the primordial genetic material is fragmentary at best. The chemical replication of oligonucleotides long enough to perform catalytic functions is particularly problematic because of the low efficiency of nonenzymatic template copying. Here we show that this problem can be circumvented by assembling a functional ribozyme by the templated ligation of short oligonucleotides. However, this approach creates a new problem because the splint oligonucleotides used to drive ribozyme assembly strongly inhibit the resulting ribozyme. We explored three approaches to the design of splint oligonucleotides that enable efficient ligation but which allow the assembled ribozyme to remain active. DNA splints, splints with G:U wobble pairs, and splints with G to I (Inosine) substitutions all allowed for the efficient assembly of an active ribozyme ligase. Our work demonstrates the possibility of a transition from nonenzymatic ligation to enzymatic ligation and reveals the importance of avoiding ribozyme inhibition by complementary oligonucleotides.