Histone acetyltransferases (HATs) are key players in epigenetic regulation of gene function. Recent discovery of diverse HAT substrates implicates a broad spectrum of cellular functions of HATs. Many pathological processes are also intimately associated with dysregulation of HAT levels and activities. However, detection of enzymatic activity of HATs has been challenging and significantly impeded drug discovery. To advance the field, we developed a convenient one-pot mix-and-read strategy that is capable to directly detect the acylated histone product via fluorescent readout. The strategy integrated three technological platforms, bioorthogonal HAT substrate labeling, alkyne-azide click chemistry, and quenching-FRET, into one system for effective probing of HAT enzyme activity.
Bioorthogonal chemical probe; protein acetylation; HAT activity; click reaction; Quenching-FRET
Quorum sensing is cell-to-cell communication that allows bacteria to coordinate attacks on their host by inducing virulent gene expression, biofilm production, and other cellular functions, including antibiotic resistance. AHL synthase enzymes synthesize N-acyl-L-homoserine lactones, commonly referred to as autoinducers, to facilitate quorum sensing in Gram-negative bacteria. Studying the synthases, however, has shown to be a difficult road. Two assays including a radiolabel and a colorimetric (DCPIP) assay are well documented in literature to study AHL synthases. In this paper, we describe additional methods that include an HPLC-based, C-S bond cleavage, and coupled assays to investigate this class of enzymes. In addition, we compare and contrast each assay for both acyl-CoA and acyl-ACP utilizing synthases. The expanded toolkit described in this study should facilitate mechanistic studies on quorum sensing signal synthases and expedite discovery of antivirulent compounds.
Enzymologist Tool Box
The N-acyl-homoserine lactone synthases are attractive targets to control virulence in Gram-negative bacteria. However, robust assays required to study these enzymes are fairly limited. Here, we have expanded the assay tool kit to expedite mechanistic studies and inhibitor discovery for this important class of enzymes.
quorum sensing; acyl-homoserine lactone synthase; enzyme assay; methylthioadenosine nucleosidase; xanthine oxidase; Michaelis constant; S-adenosyl-L-methionine; S-adenosyl-L-homocysteine; acyl-CoA; acyl-ACP
Real-time tracking of RNA expression can provide insight into the mechanisms used to generate cellular diversity, as well as help determine the underlying causes of disease. Here we present the exploration of azide-modified nucleoside analogues and their ability to be metabolically incorporated into cellular RNA. We report robust incorporation of adenosine analogues bearing azide handles at both the 2′- and N6-positions; 5-methylazidouridine was not incorporated into cellular RNA. We further demonstrate selectivity of our adenosine analogues for transcription and polyadenylation. We predict that azidonucleosides will find widespread utility in examining RNA functions inside living cells, as well as in more complex systems such as tissues and living animals.
Azide Functionality, Inc. We explored Functionality into Cellular RNA azidonucleoside incorporation into cellular RNA, revealing selectivity for azidoadenosine analogues and no incorporation of 5-azidouridine. This expansion of the bioorthogonal toolkit for RNA will further investigation into RNA expression and processing and provide a platform for analyzing the growing list of RNA functions beyond protein encoding.
azides; imaging; modified nucleosides; RNA incorporation; transcription
The increasing number of enzyme applications in chemical synthesis calls for new engineering methods to develop the biocatalysts of the future. An interesting concept in enzyme engineering is the generation of large‐scale mutational data in order to chart protein mutability landscapes. These landscapes allow the important discrimination between beneficial mutations and those that are neutral or detrimental, thus providing detailed insight into sequence–function relationships. As such, mutability landscapes are a powerful tool with which to identify functional hotspots at any place in the amino acid sequence of an enzyme. These hotspots can be used as targets for combinatorial mutagenesis to yield superior enzymes with improved catalytic properties, stability, or even new enzymatic activities. The generation of mutability landscapes for multiple properties of one enzyme provides the exciting opportunity to select mutations that are beneficial either for one or for several of these properties. This review presents an overview of the recent advances in the construction of mutability landscapes and discusses their importance for enzyme engineering.
biocatalysis; enzyme engineering; hotspots; mutability landscapes; mutagenesis
The Euphorbiaceae produce a wide variety of bioactive diterpenoids. These include the lathyranes, which have received much interest due to their ability to inhibit the ABC transporters responsible for the loss of efficacy of many chemotherapy drugs. The lathyranes are also intermediates in the biosynthesis of range of other bioactive diterpenoids with potential applications in the treatment of pain, HIV and cancer. We report here a gene cluster from Jatropha curcas that contains the genes required to convert geranylgeranyl pyrophosphate into a number of diterpenoids, including the lathyranes jolkinol C and epi‐jolkinol C. The conversion of casbene to the lathyranes involves an intramolecular carbon–carbon ring closure. This requires the activity of two cytochrome P450s that we propose form a 6‐hydroxy‐5,9‐diketocasbene intermediate, which then undergoes an aldol reaction. The discovery of the P450 genes required to convert casbene to lathyranes will allow the scalable heterologous production of these potential anticancer drugs, which can often only be sourced in limited quantities from their native plant.
casbene; cytochromes; diterpenoids; gene clusters; lathyranes
Sirtuins (SIRTs) are a family of NAD+‐dependent histone deacetylases. In mammals, dysfunction of SIRTs is associated with age‐related metabolic diseases and cancers, so SIRT modulators are considered attractive therapeutic targets. However, current screening methodologies are problematic, and no tools for imaging endogenous SIRT activity in living cells have been available until now. In this work we present a series of simple and highly sensitive new SIRT activity probes. Fluorescence of these probes is activated by SIRT‐mediated hydrolytic release of a 4‐(4‐dimethylaminophenylazo)benzoyl (Dabcyl)‐based FRET quencher moiety from the ϵ‐amino group of lysine in a nonapeptide derived from histone H3K9 and bearing a C‐terminal fluorophore. The probe SFP3 detected activities of SIRT1, ‐2, ‐3, and ‐6, which exhibit deacylase activities towards long‐chain fatty acyl groups. We then truncated the molecular structure of SFP3 in order to improve both its stability to peptidases and its membrane permeability, and developed probe KST‐F, which showed specificity for SIRT1 over SIRT2 and SIRT3. We show that KST‐F can visualize endogenous SIRT1 activity in living cells.
enzymatic reaction; fluorescent probes; FRET; imaging agents; quencher dyes; sirtuins
Gene expression is extensively regulated by the occurrence and distribution of the epigenetic marker 2′‐deoxy 5‐methylcytosine (5mC) in genomic DNA. Because of its effects on tumorigenesis there is an important link to human health. In addition, detection of 5mC can serve as an outstanding biomarker for diagnostics as well as for disease therapy. Our previous studies have already shown that, by processing O
6‐alkylated 2′‐deoxyguanosine triphosphate (dGTP) analogues, DNA polymerases are able to sense the presence of a single 5mC unit in a template. Here we present the synthesis and evaluation of an extended toolbox of 6‐substituted 2‐aminopurine‐2′‐deoxyribonucleoside 5′‐triphosphates modified at position 6 with various functionalities. We found that sensing of 5‐methylation by this class of nucleotides is more general, not being restricted to O
6‐alkyl modification of dGTP but also applying to other functionalities.
5mC; DNA methylation; DNA polymerases; epigenetics; gene expression; modified nucleotides
The CRISPR‐Cas9 gene editing system requires Cas9 endonuclease and guide RNAs (either the natural dual RNA consisting of crRNA and tracrRNA or a chimeric single guide RNA) that direct site‐specific double‐stranded DNA cleavage. This communication describes a click ligation approach that uses alkyne–azide cycloaddition to generate a triazole‐linked single guide RNA (sgRNA). The conjugated sgRNA shows efficient and comparable genome editing activity to natural dual RNA and unmodified sgRNA constructs.
conjugation; CRISPR-Cas9; gene technology; oligonucleotides; single guide RNA
Systematic alanine scanning of the linear peptide bisebromoamide (BBA), isolated from a marine cyanobacterium, was enabled by solid‐phase peptide synthesis of thiazole analogues. The analogues have comparable cytotoxicity (nanomolar) to that of BBA, and cellular morphology assays indicated that they target the actin cytoskeleton. Pathway inhibition in human colon tumour (HCT116) cells was explored by reverse phase protein array (RPPA) analysis, which showed a dose‐dependent response in IRS‐1 expression. Alanine scanning reveals a structural dependence to the cytotoxicity, actin targeting and pathway inhibition, and allows a new readily synthesised lead to be proposed.
alanine scan; cell morphology; nonribosomal peptides; reverse phase protein array; solid-phase synthesis; structure–activity relationships
Mutations and post‐translational modifications of amyloid‐β (Aβ) peptide in its N terminus have been shown to increase fibril formation, yet the molecular mechanism is not clear. Here we investigated the kinetics of the interactions of copper with two Aβ peptides containing Familial Alzheimer's disease (FAD) mutations (English (H6R) and Tottori (D7N)), as well as with Aβ peptide phosphorylated at serine 8 (pS8). All three peptides bind to copper with a similar rate as the wild‐type (wt). The dissociation rates follow the order pS8>H6R>wt>D7N; the interconversion between the two coordinating species occurs 50 % faster for H6R and pS8, whereas D7N had only a negligible effect. Interestingly, the rate of ternary complex (copper‐bridged heterodimer) formation for the modified peptides was significantly faster than that for wt, thus leading us to propose that FAD and sporadic AD might share a kinetic origin for the enhanced oligomerisation of Aβ.
amyloid beta-peptides; copper; fluorescence spectroscopy; kinetics; reaction mechanism
Macrocyclization can be used to constrain peptides in their bioactive conformations, thereby supporting target affinity and bioactivity. In particular, for the targeting of challenging protein–protein interactions, macrocyclic peptides have proven to be very useful. Available approaches focus on the stabilization of α‐helices, which limits their general applicability. Here we report for the first time on the use of ring‐closing alkyne metathesis for the stabilization of an irregular peptide secondary structure. A small library of alkyne‐crosslinked peptides provided a number of derivatives with improved target affinity relative to the linear parent peptide. In addition, we report the crystal structure of the highest‐affinity derivative in a complex with its protein target 14‐3‐3ζ. It can be expected that the alkyne‐based macrocyclization of irregular binding epitopes should give rise to new scaffolds suitable for targeting of currently intractable proteins.
macrocyclization; peptide secondary structures; peptidomimetics; protein–protein interactions; ring-closing alkyne metathesis
Engineered enzyme variants of potato epoxide hydrolase (StEH1) display varying degrees of enrichment of (2R)‐3‐phenylpropane‐1,2‐diol from racemic benzyloxirane. Curiously, the observed increase in the enantiomeric excess of the (R)‐diol is not only a consequence of changes in enantioselectivity for the preferred epoxide enantiomer, but also to changes in the regioselectivity of the epoxide ring opening of (S)‐benzyloxirane. In order to probe the structural origin of these differences in substrate selectivity and catalytic regiopreference, we solved the crystal structures for the evolved StEH1 variants. We used these structures as a starting point for molecular docking studies of the epoxide enantiomers into the respective active sites. Interestingly, despite the simplicity of our docking analysis, the apparent preferred binding modes appear to rationalize the experimentally determined regioselectivities. The analysis also identifies an active site residue (F33) as a potentially important interaction partner, a role that could explain the high conservation of this residue during evolution. Overall, our experimental, structural, and computational studies provide snapshots into the evolution of enantioconvergence in StEH1‐catalyzed epoxide hydrolysis.
enantioselectivity; epoxide hydrolysis; evolutionary snapshots; laboratory evolution; protein engineering
Fluorogenic enzyme probes go from a dark to a bright state following hydrolysis and can provide a sensitive, real-time readout of enzyme activity. They are useful for examining enzymatic activity in bacteria, including the human pathogen Mycobacterium tuberculosis. Herein, we describe two fluorogenic esterase probes derived from the far-red fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). These probes offer enhanced optical properties compared to existing esterase probes because the hydrolysis product, DDAO, excites above 600 nm while retaining a good quantum yield (ϕ=0.40). We validated both probes with a panel of commercially available enzymes alongside known resorufin- and fluorescein-derived esterase substrates. Furthermore, we used these probes to reveal esterase activity in protein gel-resolved mycobacterial lysates. These probes represent new tools for esterase detection and characterization and should find use in a variety of applications.
esterases; fluorescence; fluorogenic probes; lipases; tuberculosis
Oxygen-to-sulfur substitutions in DNA phosphate often enhance affinity for DNA-binding proteins. Our previous studies have suggested that this effect of sulfur substitution of both OP1 and OP2 atoms is due to an entropic gain associated with enhanced ion-pair dynamics. In this work, we studied stereospecific effects of single sulfur substitution of either the OP1 or OP2 atom in DNA phosphate at the Lys57 interaction site of the Antennapedia homeodomain-DNA complex. By crystallography, we obtained the structural information on the RP and SP diastereomers of the phosphoromonothioate and their interaction with Lys57. By fluorescence-based assays, we found significant affinity enhancement upon sulfur substitution of the OP2 atom. By NMR spectroscopy, we found significant mobilization of the Lys57 side-chain NH3+ group upon sulfur substitution of the OP2 atom. These data provide further mechanistic insight into the affinity enhancement by oxygen-to-sulfur substitution in DNA phosphate.
Oxygen-to-sulfur substitution in DNA phosphate can enhance protein-DNA binding affinity. To gain mechanistic insight into this phenomenon, we compared the impacts of 3 different types of phosphorothioation on binding affinity, ion-pair dynamics, and structure.
Dynamics; Ion pair; NMR spectroscopy; Phosphorothioate; Protein-DNA interaction
We describe the genetically directed incorporation of aminooxy functionality into recombinant proteins by using a mutant Methanosarcina barkeri pyrrolysyl‐tRNA synthetase/tRNACUA pair. This allows the general production of nonhydrolysable ubiquitin conjugates of recombinant origin by bioorthogonal oxime ligation. This was exemplified by the preparation of nonhydrolysable versions of diubiquitin, polymeric ubiquitin chains and ubiquitylated SUMO. The conjugates exhibited unrivalled isostery with the native isopeptide bond, as inferred from structural and biophysical characterisation. Furthermore, the conjugates functioned as nanomolar inhibitors of deubiquitylating enzymes and were recognised by linkage‐specific antibodies. This technology should provide a versatile platform for the development of powerful tools for studying deubiquitylating enzymes and for elucidating the cellular roles of diverse polyubiquitin linkages.
genetic code expansion; isopeptide; oxime; polymerization; synthetic methods; ubiquitylation
Pseudomonas aeruginosa uses N-acylated L-homoserine lactone signals and a triumvirate of LuxR-type receptor proteins – LasR, RhlR, and QscR – for quorum sensing (QS). Each of these receptors can contribute to QS activation or repression, and thereby, the control of myriad virulence phenotypes in this pathogen. LasR has traditionally been considered at the top of the QS receptor hierarchy in P. aeruginosa; however, recent reports suggest that RhlR plays a more prominent role in infection than originally predicted, in some circumstances superseding LasR. Herein, we report the characterization of a set of synthetic, small molecule agonists and antagonists of RhlR. Using E. coli reporter strains, we demonstrate that many of these compounds can selectively activate or inhibit RhlR instead of LasR and QscR. Moreover, several molecules maintain their activities in P. aeruginosa at concentrations analogous to native RhlR-signal levels. These compounds represent useful chemical probes to study the role of RhlR in the complex QS circuitry of P. aeruginosa, its direct (and indirect) effects on virulence, and its overall merit as a target for anti-infective therapy.
N-acylated L-homoserine lactone; LuxR-type receptor; RhlR; virulence; quorum sensing
Understanding enzymatic Diels—Alder (DA) reactions that can form complex natural product scaffold is of considerable interest. Sch 210972 1, a potential anti-HIV fungal natural product, contains a decalin core that is proposed to form via a DA reaction. We identified the gene cluster responsible for the biosynthesis of 1 and heterologously reconstituted the biosynthetic pathway in Aspergillus nidulans to characterize the enzymes involved. Most notably, deletion of cghA resulted in a loss of stereoselective decalin core formation, yielding both an endo
1 and a diastereomeric exo adducts of the proposed DA reaction. Complementation with cghA restored the sole formation of 1. Density functional theory computation of the proposed DA reaction provided a plausible explanation of the observed pattern of product formation. Based on our study, we propose that lipocalin-like CghA is responsible for the stereoselective intramolecular [4+2] cycloaddition that forms the decalin core of 1.
cycloaddition; decalin natural product; density functional calculations; fungal metabolite; tetramic acid
Many proteins suffer from sub-optimal pharmacokinetics (PK) that limit their utility as drugs. The efficient synthesis of polymer conjugates of protein drugs with tunable PK to optimize their in vivo efficacy is hence critical. We report here the first study of the in vivo behavior of a site-specific conjugate of a zwitterionic polymer and a protein. To synthesize the conjugate, we first installed an initiator for atom transfer radical polymerization (ATRP) at the N-terminus of myoglobin (Mb-N-Br). Subsequently, in situ ATRP was carried out in aqueous buffer to grow an amine-functionalized polymer from Mb-N-Br. The cationic polymer was further derivatized to two zwitterionic polymers by reaction of the amine groups of the cationic polymer with iodoacetic acid to obtain poly(carboxybetaine methylacrylate) with a 1 carbon spacer (C1) (PCBMA), and sequentially with 3-iodopropionic acid and iodoacetic acid to obtain PCBMA(mix) with a mixture of 1 carbon (C1) and 2 carbon (C2) spacer. The Mb-N-PCBMA polymer conjugates had a longer in vivo plasma half-life than a PEG-like comb polymer conjugate of similar MW. The structure of the zwitterion plays a role in controlling the in vivo behavior of the conjugate, as the PCBMA conjugate with a C1 spacer had significantly longer plasma circulation than the conjugate with a mixture of C1 and C2 spacers.
Zwitterionic; Site-specific modification; Protein-polymer conjugate; ATRP; Pharmacokinetics
Iterative type I polyketide synthases (PKSs) from fungi are multifunctional enzymes that use their active sites repeatedly in a highly ordered sequence to assemble complex natural products. A phytotoxic macrolide with anticancer properties, 10,11-dehydrocurvularin (DHC), is produced by cooperation of a highly reducing (HR) iterative PKS and a non-reducing (NR) iterative PKS. We have identified the DHC gene cluster in Alternaria cinerariae, heterologously expressed the active HR PKS (Dhc3) and NR PKS (Dhc5) in yeast and compared them to corresponding proteins that make DHC in Aspergillus terreus. Phylogenetic analysis, and homology modeling of these enzymes has identified variable surfaces and conserved motifs that are implicated in product formation.
We have identified and heterologous expressed two iterative type I PKSs necessary for production of the phytotoxic anticancer agent 10,11-dehydrocurvularin (DHC) in Alternaria cinerariae. This afforded the unique opportunity for bioinformatic comparison to the analogous metabolite gene cluster in Aspergillus terreus, which highlighted key structural features necessary for metabolite production.
bioinformatics; biosynthesis; dehydrocurvularin; heterologous expression; polyketide
We present the first crystallographic insight into the interactions of an ionic liquid (IL) with an enzyme, which has widespread implications for stabilizing enzymes in IL media for biocatalysis. Structures of Bacillus subtilis lipase A (lipA) and an IL stable varaint (QM-lipA) were obtained in the presence of increasing concentrations of 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). These studies revealed that the [BMIM] cation interacts to surface residues via hydrophobic and cation-π interactions. Of specific interest was the disruption of internal stacking interactions of aromatic side chains by [BMIM], which provide structural evidence for the mechanism of enzyme denaturation by ILs. The interaction with [BMIM] and Cl ions to lipA was reduced by the stabilizing mutations Y49E and G158E in QM-lipA. Ultimately, these findings present the molecular basis for stabilizing enzymes from IL-induced inactivation as well as the selection of ILs that are less denaturing.
The nature of the interaction of ionic liquids with enzymes was investigated by determining the crystal structure of Bacillus subtilis lipase A and an ionic liquid stable variant in the presence of increasing concentrations of [BMIM][Cl]. Analysis of the binding modes of the ionic liquid offer unprecedented insight into the molecular basis for stabilizing enzymes from ionic liquid-induced denaturation.
Lipase; Ionic Liquids; 1-Butyl-3-methylimidazolium chloride; Biocatalysis; Crystallography
Eugenol oxidase (EUGO) from Rhodococcus jostii RHA1 had previously been shown to convert only a limited set of phenolic compounds. In this study, we have explored the biocatalytic potential of this flavoprotein oxidase, resulting in a broadened substrate scope and a deeper insight into its structural properties. In addition to the oxidation of vanillyl alcohol and the hydroxylation of eugenol, EUGO can efficiently catalyze the dehydrogenation of various phenolic ketones and the selective oxidation of a racemic secondary alcohol—4‐(1‐hydroxyethyl)‐2‐methoxyphenol. EUGO was also found to perform the kinetic resolution of a racemic secondary alcohol. Crystal structures of the enzyme in complexes with isoeugenol, coniferyl alcohol, vanillin, and benzoate have been determined. The catalytic center is a remarkable solvent‐inaccessible cavity on the si side of the flavin cofactor. Structural comparison with vanillyl alcohol oxidase from Penicillium simplicissimum highlights a few localized changes that correlate with the selectivity of EUGO for phenolic substrates bearing relatively small p‐substituents while tolerating o‐methoxy substituents.
biocatalysis; dehydrogenation; enzyme structures; kinetic resolution; oxidases
The application of commonly used force spectroscopy in biological systems is often limited by the need for an invasive tether connecting the molecules of interest to a bead or cantilever tip. Here we present a DNA origami‐based prototype in a comparative binding assay. It has the advantage of in situ readout without any physical connection to the macroscopic world. The seesaw‐like structure has a lever that is able to move freely relative to its base. Binding partners on each side force the structure into discrete and distinguishable conformations. Model experiments with competing DNA hybridisation reactions yielded a drastic shift towards the conformation with the stronger binding interaction. With reference DNA duplexes of tuneable length on one side, this device can be used to measure ligand interactions in comparative assays.
DNA hybridisation; DNA origami; DNA structures; self-assembly; sensors
Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis–Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N′‐Boc‐protected 5‐carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high‐throughput manner.
deubiquitinating enzymes; FRET; native chemical ligation; solid-phase synthesis; ubiquitin conjugates
Flavin‐dependent halogenases are potentially useful biocatalysts for the regioselective halogenation of aromatic compounds. Haloaromatic compounds can be utilised in the synthesis and biosynthesis of pharmaceuticals and other valuable products. Here we report the first X‐ray crystal structure of a tryptophan 6‐halogenase (SttH), which enabled key residues that contribute to the regioselectivity in tryptophan halogenases to be identified. Structure‐guided mutagenesis resulted in a triple mutant (L460F/P461E/P462T) that exhibited a complete switch in regioselectivity; with the substrate 3‐indolepropionate 75 % 5‐chlorination was observed with the mutant in comparison to 90 % 6‐chlorination for the wild‐type SttH. This is the first clear example of how regiocomplementary halogenases can be created from a single parent enzyme. The biocatalytic repertoire of SttH was also expanded to include a range of indolic and non‐indolic substrates.
aryl halides; biocatalysis; halogenation; mutagenesis; regioselectivity