ChemBioChem

Microbial transglutaminase (MTG) has shown to be a powerful biocatalytic glue for site-specific crosslinking of a range of biomolecules and synthetic molecules, those handled with an MTG-reactive moiety. The preparation of active recombinant MTG requires the posttranslational proteolytic digestion of propeptide working as an intramolecular chaperon to assist the correct folding of MTG zymogen (MTGz) in the biosynthesis. Herein, we propose an engineered active zymogen of MTG (EzMTG) that is expressed as soluble form in the host E. coli cytosol and exhibits the cross-linking activity without limited proteolysis. Based on the 3D structure of MTGz and serendipitous findings, saturated mutagenesis of K10 or Y12 in propeptide domain leads to generate several active MTGz mutants. In particular, K10D/Y12G mutant exhibited the catalytic activity comparable with a mature form. However, the expression level was low possibly due to the reduction of chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. By contrast, soluble K10R/Y12A mutant is expressed in the cytosol of host E. coli and exhibited unique substrate-dependent reactivity toward peptidyl substrates. The quantitative analysis of the binding affinity of mutated propeptide to the active site suggested the trade-off relationship of EzMTGs between the binding affinity and the catalytic activity. Our proof-of-concept study provides insights into the design of a new biocatalyst by using the zymogen as a scaffold and will convey a potential route to the high-throughput screening of MTG mutants for bioconjugation applications.

Cyclodipeptide synthases (CDPSs) use aminoacylated tRNAs to produce cyclic dipeptide natural products which can have anticancer and neuroprotective activity. Despite their potential, applications involving CDPSs are hindered by enzyme instability and challenges in producing aminoacylated tRNAs. Immobilizing enzymes can enhance stability and recyclability, yet studies on immobilized enzymes using aminoacylated tRNAs are lacking. Here, we immobilized the CDPS enzyme from Parcubacteria bacterium RAAC4_OD1_1 (PbCDPS) using three sustainable supports: biochar from waste materials, calcium-alginate beads, and chitosan beads. Active PbCDPS immobilization led to production of the cyclodipeptide cyclo (His-Glu) (cHE). Notably, following activation with glutaraldehyde, a five-fold increase in cHE production was observed, while the immobilized enzyme remained active for seven consecutive cycles. Furthermore, we co-immobilized three enzymes required for the cascade reaction yielding cHE, all of which require aminoacyl-tRNA substrates (PbCDPS, histidyl-tRNA synthetase, and glutamyl-tRNA synthetase). This enzymatic cascade successfully generated the cyclic dipeptide of interest, showcasing the potential of immobilizing complex enzymes operating in cascade on a single support. We demonstrated that tRNAs remained free in solution without adsorption onto beads. This work paves the way for the immobilization of enzymes utilizing tRNAs and potentially other complex substrates, expanding the spectrum of reactions exploitable with this technology. Graphical AbstractO_LIFor Table of Contents Only C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/604810v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@12794baorg.highwire.dtl.DTLVardef@1956c09org.highwire.dtl.DTLVardef@e7f800org.highwire.dtl.DTLVardef@14ac97d_HPS_FORMAT_FIGEXP M_FIG C_FIG

VdtB, the multiple-copper oxidase (MCO) from the bisnaphthopyrone (M)-viriditoxin biosynthetic pathway in Paecilomyces variotii, was shown to catalyze regioselective 6,6'-coupling of semi-viriditoxin (1). The stereoselectivity of the oxidative coupling reaction for the production of the atropisomer (M)-viriditoxin, however, was controlled by VdtD, a non-catalytic dirigent protein from the pathway. In this work, VdtB either alone or together with VdtD were investigated for its stereoselective control upon coupling of other monomeric naphthopyrone derivatives from the pathway with different minor structural variations in terms of presence/absence of O-methylation at C7-position and C3-C4{Delta} 2 double bond on the pyrone ring, and the different side-chain modifications. We showed that VdtB could favour either M- or P-form coupling in a substrate-dependent manner. For some substrates, VdtB could catalyze oxidative coupling in an enantiomerically selective manner. The efficiency of the VdtD in exerting stereoselective control of the oxidative coupling reaction also varies between substrates. The results point to a model whereby VdtB and VdtD form a VdtB-ligand-VdtD complex in which the stereochemical outcome of the coupling reaction depends on how the substrate interacts with both proteins, based on the substrate structure. Our findings contributed to a more comprehensive understanding of dirigent protein-mediated MCO-catalyzed stereoselective oxidative coupling reactions in fungi.

Enzymes play a pivotal role in "green chemistry" as tools for biocatalysis. Oxidoreductase enzymes are especially useful for carrying out key electron transfer (redox) steps towards a wide range of chemical transformations (e.g., asymmetric hydrogenation, oxygenation, hydroxylation, epoxidation, or Baeyer-Villiger oxidation) that might not otherwise be available to chemists through conventional (nonbiological) synthetic approaches. The ability to screen oxidoreductase activity is important in identifying useful biocatalysts from nature, and also towards engineering novel ones through directed evolution. Many valuable redox enzymes are dependent upon NAD(P)H as an electron donating co-substrate (or conversely, upon NAD(P)+ as an electron acceptor), and the common method to detect their activity is to monitor the change in absorbance at 340 nm as NAD(P)H is converted to NAD(P)+ (or vice versa). The limited sensitivity of this method presents a challenge in detecting very low levels of oxidoreductase activity, and this can prove very difficult to begin engineering enzymes as improved biocatalysts when the rates of natural enzymes may be slow for a desired redox reaction. Herein, we report a fluorescence-based, enzyme cascade-coupled system that we have developed to detect oxidoreductase activity with orders of magnitude more sensitivity than conventional absorbance-based assays. While recycling NAD(P)H from NAD(P)+, the coupled enzyme cascade triggers cleavage of a fluorogenically labeled probe, releasing a strong fluorescent signal. This allows detection of very low levels of a specific oxidoreductase activity that we may wish to magnify by directed evolution using our assay in high-throughput screening.

Developing artificial enzymes is challenging because it requires precise design of active sites with well-arranged amino acid residues. Histidine-rich oligopeptides have been recently shown to exhibit peroxidase-mimetic activities, but their catalytic function relies on maintaining unique supramolecular structures. This work demonstrates the design of a specific array of histidine residues on the internal surface of the ferritin cage to function as an active center for catalysis. The crystal structures of the ferritin mutants revealed histidine-histidine interactions, forming well-defined histidine clusters (His-clusters). These mutants exhibit peroxidase-mimetic activities by oxidizing 3,3,5, 5-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide. Molecular dynamics simulations further highlight the co-localization of TMB and hydrogen peroxide at the histidine-rich clusters, indicating that the confined environment of the ferritin cage enhances their interactions. This study presents a simple yet effective approach to design cofactor-free artificial enzymes, paving the way for innovations in bioinspired catalysis.

G-quadruplex (G4) structures have emerged as singular therapeutic targets for cancer and neurodegeneration. Autophagy is a housekeeping cellular homeostatic mechanism and deregulation of autophagy is common in cancer and in neurodegenerative diseases. In this study, we identified the presence of 46 putative G4 sequences in the MTOR gene by use of QGRS mapper tool. We sought to connect these putative G4 sequences to a functional context by leveraging G4-targeting ligands. A G4-selective dimeric carbocyanine dye Bis-4,3 and the porphyrin TMPyP4 were used to affect the replication, transcription and translation of the MTOR gene. The ligand-induced induction of autophagic pathway via MTOR gene regulation was monitored upon treatment of HeLa and SHSY-5Y cells with G4-targeting ligands. The use of Bis-4,3 was compared with the known G4-stabilizing activity of TMPyP4. Our results show that treatment with G4-selective ligands downregulates mTOR activity and leads to the induction of excessive autophagy. This is first report on effect of G4-selective ligands on MTOR regulation and mTOR expression. mTOR being the key negative regulator of autophagy, the current work suggests potential of G4 stabilizing ligands towards induction of autophagy through the downregulation of mTOR.

HMGB1 (high-mobility group box 1) protein is a nonhistone chromatin-associated protein that has been widely reported to be a representative damage-associated molecular pattern (DAMP) and to play a pivotal role in proinflammatory process once it is in an extracellular location. Accumulating evidence has shown HMGB1 undergoes extensive PTMs that remarkably regulated its conformation, localization, and intermolecular interaction. However, the PTMrelated study has been dramatically hindered by the difficulty to access to homogenous proteins with site-specific PTMs of interest. Here, we introduce a protein semi-synthesis strategy via salicylaldehyde ester-mediated chemical ligations (Ser/Thr ligation and Cys/Pen ligation, STL/CPL). This methodology has enabled us to generate N-terminal acetylated HMGB1 proteins in high purity. Further studies revealed that the acetylation on N-terminus regulates its interaction with heparin and modulates its stability, representing a regulatory switch to control the HMGB1s activity.

Signal transduction through sealed biological membranes is among the most important evolutionary achievements. Herein, we focus on the development of artificial signal transduction mechanisms and engineer a bionic receptor with capacity of transduction of biological signals across biological membranes using tools of chemistry. The bionic receptor described in this work exhibits similarity with the natural counterpart in the most essential characteristics: in having an exofacial ligand for signal capture, in being membrane anchored, and in featuring a releasable secondary messenger molecule, which performs enzyme activation in the endo volume. The main difference with the natural receptors is that signal transduction across the lipid bilayer was performed using the tools of organic chemistry, namely a self-immolative linker. The highest novelty of our work is that the artificial signalling cascade designed herein achieved transmembrane activation of enzymatic activity, as is the hallmark of activity by natural signalling receptors.

We present polysaccharide-based nanoparticles able to associate and increase the catalytic activity of the maltose-binding MBP317-347 switch enzyme. Fluorescence quenching and molecular docking studies along with the partial resistance to increasing pH and ionic strength indicate that the increase in enzymatic activity is due to a specific interaction between the maltose binding pocket on MBP317-347 and alginate exposed on the surface of the nanoparticles. Finally, we show that the hybrid self co-assembled particles increase the half-life of MBP317-347 over six-fold at 37{degrees}C, thus reflecting their potential use as a macromolecular drug delivery system.

Engineering of the natural light-regulated transcription factors for the purposeful generation of synthetic photo-switches is a widely adopted strategy in synthetic biology and optogenetics. Aureochrome is one such potential, naturally occurring photoreceptor-cum-transcription factor that can be used for the development of synthetic optogenetic tools, especially meant for light-regulated gene expression. It is however well-known that different biological events demand different duration of signaling state lifetime and diverse affinity towards different DNA substrates. Therefore, in this study, we produce multiple variants of aureochromes via random mutagenesis - mimicking single generation directed evolution. After screening the single generation variants for in-frame transcripts, we sort them on the basis of altered photocycle kinetics as well as DNA-binding affinity. The variants that exhibited reversible photochemistry and light-regulated DNA-binding ability similar/comparable to that of the wild-type despite incurring mutations, are characterized and discussed in this manuscript. These can later be subjected to successive rounds of random mutations to get an eventual superior variety with enhanced functionality. Even the apparently unsuccessful ones, which depicted drastic alteration of photochemical/DNA-binding properties, helped us to identify amino acid residues - lesser known to be indispensable for a certain biological activity. The process of diversification and selection via random mutagenesis not only explains the functional significance of the different amino acids in aureochromes but also generates a repertoire of appropriate aureochrome variants that may be used in optogenetics.

Molecular switching plays a critical role in biological and displaying systems. Here we demonstrate the first use of peptides to operate molecular switches of donor-acceptor Stenhouse adducts (DASAs), a series of negative photochromes that are highly promising for applications ranging from smart material to biological systems. Fluorescence imaging proved A{beta}40 species could make SHA-2 more stable in the linear configuration than without peptide and decrease the rate of molecular switching. According to molecular dynamics simulation, SHA-2 bound to protein resulted in substantial changes in the tertiary structure of A{beta}40 monomer with the region of Glu22-Ala30 partially unfolded and being more exposed to water. This structural change is likely to impede the aggregation of A{beta}40, as evidenced by fluorescence and ProteoStat(R) aggresome detection experiments. SHA-2 is able to inhibit the aggregation of A{beta}40 by producing the off-pathway structures. These results open ample opportunities for optically addressable potential widely apply DASAs in the biological system based on this peptides-tailor process.

Hydrolases are important molecules that are involved in a wide range of biological functions and their activities are tightly regulated in healthy or diseased states. Detecting or imaging the activities of hydrolases, therefore, can reveal underlying molecular mechanisms in the context of cells to organisms, and their correlation with different physiological conditions can therefore be used in diagnosis. Due to the nature of hydrolases, substrate-based probes can be activated in their catalytic cycles, and cleavage of covalent bonds frees reporter moieties. For test-tube type bulk detection, spatial resolution is not a measure of importance, but for cell- or organism-based detection or imaging, spatial resolution is a key factor for probe sensitivity that influences signal-to-background ratio. One strategy to improve spatial resolution of the probes is to form a covalent linkage between the reporter moiety and intracellular proteins upon probe activation by the enzyme. In this work, we developed a generalizable linker chemistry that would allow in situ labeling of various imaging moieties via quinone methide species. To do so, we synthesized probes containing a monofluoromethyl or a difluoromethyl groups for {beta}-galactosidase activation, while using fluorescein as a fluorescent reporter. The labeling efficacy of these two probes was evaluated in vitro. The probe bearing a monofluormethyl group exhibited superior labeling efficiency in imaging {beta}-galactosidase activity in living cells. This study provides a versatile linker for applying quinone methide chemistry in the development of hydrolase-targeting probes involving in situ labeling.

Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in their proximity via distance dependent processes such as Forster resonance energy transfer (FRET). Many FPs have a tendency to oligomerise, which is likely to be promoted through attachment to associating proteins through increases in local FP concentration. We show here that on association of FP pairs, the inherent function of the FPs can alter. Artificial dimers were constructed using a bioorthogonal Click chemistry approach that combined a commonly used green fluorescent protein (superfolder GFP) with itself, a yellow FP (Venus) or a red FP (mCherry). In each case dimerisation changes the inherent fluorescent properties, including FRET capability. The GFP homodimer demonstrated synergistic behaviour with the dimer being brighter than the sum of the two monomers. The structure of the GFP homodimer revealed that a water-rich interface is formed between the two monomers, with the chromophores being in close proximity with favourable transition dipole alignments. Dimerisation of GFP with Venus results in a complex displaying [~]86% FRET efficiency, which is significantly below the near 100% efficiency predicted. When GFP is complexed with mCherry, FRET and mCherry fluorescence itself is essentially lost. Thus, the simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein-protein interactions promote FP interaction. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/838888v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1863e41org.highwire.dtl.DTLVardef@61255dorg.highwire.dtl.DTLVardef@1dc188forg.highwire.dtl.DTLVardef@d066ad_HPS_FORMAT_FIGEXP M_FIG C_FIG

Understanding the mechanism by which Artemisinin conjugated with phototheranostic probes for reducing side effects (AC-Pr-RSE) will guide endeavor to improve the therapeutic outcomes. Herein, the first report describes the application of a cytotoxic artemisinin derivative and N-methyliminodiacetic acid (MIDA) boronates conjugated with a naphthalimide fluorophore for the detection of H2O2 in the Golgi apparatus. A comparative analysis of the localization signals from the fluorescent artemisinin derivative and organelle-specific dyes uncovered that the Golgi apparatus functions as the primary site of its accumulation, which could efficiently enhance the intracellular reactive oxygen species (ROS) level and induce cell apoptosis. This work highlights the potential of AC-Pr-RSE study, which enables us to optimize the design of theranostic probes to improve their biological activities during oxidative stress.

Hydrogels have a wide range of applications such as in biomedicine, cosmetics and soft electronics. Compared to polymer hydrogels based on covalent bonding, protein hydrogels offer distinct advantages owing to their biocompatibility and better access to molecular engineering. However, pure and natural protein hydrogels have been seldom reported except for structural proteins like collagen and silk fibrin. Here, we report the unusual ability and mechanism of a unique natural enzyme, lipoate-protein ligase A (LplA) of E. coli to self-assemble into a stimuli-responsive and reversible hydrogel of the low critical solution temperature (LCST) type. This is the first globular and catalytic protein found to form a hydrogel in response to temperature, pH and the presence of ions. Protein structure based analysis reveals the key residues responsible for the gel formation and mutational studies confirms the essential roles of hydrogen bonding between the C-terminal domains and electrostatic interactions in the N-terminal domains. Characterization of phase transitions of wild type LplA and its mutants using small angle X-ray scattering (SAXS) yields details of the gelation process from initial dimer formation over a pre-gel-state to full network development. Further electron microscopic analyses and modeling of SAXS data suggest an unusual interlinked ladder-like structure of the macroscopic crosslinking network with dimers as ladder steps. The unique features of this first reported protein hydrogel may open up hitherto inaccessible applications, especially those taking advantage of the inherent catalytic activity of LplA.

The traditional whole-cell biocatalysis typically utilizes the heterotrophic microbes as the biocatalyst, which requires carbohydrates to power the cofactor (ATP, NAD(P)H) regeneration. In this study, we sought to harness purple non-sulfur photosynthetic bacterium (PNSB) as the biocatalyst to achieve light-driven cofactor regeneration for cascade biocatalysis. We substantially improved the performance of PNSB-based biocatalysis by using a highly active and conditional expression system, blocking the side-reactions, controlling the feeding strategy, and attenuating the light shading effect. We found that 50 mM ferulic acid could be completely converted to vanillyl alcohol in the recombinant strain, reaching 7.7 g/L vanillyl alcohol. In addition, >99.9% conversion of p-coumaric acid to p-hydroxybenzoic alcohol (6.21 g/L) was similarly achieved under light-anaerobic conditions. Moreover, we examined the isoprenol utilization pathway (IUP) for pinene synthesis and 13.81 mM pinene (1.88 g/L) with 92.1% conversion rate from isoprenol was obtained. Taken together, these results suggested that PNSB could be a promising host for light-powered biotransformation, which offers an efficient approach for synthesizing value-added chemicals in a green and sustainable manner.

The search for novel synthetic tools to prepare industrial chemicals in a safer and greener manner is a continuing challenge in synthetic chemistry. In this manuscript, we report the discovery, characterization, and synthetic potential of two novel aryl-alcohol oxidases from bacteria which are able to oxidize a variety of aliphatic and aromatic alcohols in high efficiencies (up to 4970 min-1mM-1). Crystal structures revealed unusually wide-open entrance to the active-site pockets compared to that previously described for traditional fungal aryl-alcohol oxidases, which could correlate with differences in substrate scope, catalytic efficiency, and other functional properties. Preparative-scale reactions and ability to operate at high substrate loadings also demonstrate the potential of these enzymes in synthetic chemistry with turnover numbers > 30000. Moreover, their availability as soluble and active recombinant proteins enabled their use as cell-free extracts which further highlights their potential for the large-scale production of carbonyl compounds.

G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a longstanding problem in the field is how to achieve specificity amongst these different G4 structures. Here, we have developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4 Ligand conjugated Oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amounts of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

Biotechnological processes hold tremendous potential for the efficient and sustainable conversion of one-carbon (C1) substrates into complex multi-carbon products. However, the development of robust and versatile biocatalytic systems for this purpose remains a significant challenge. In this study, we report a hybrid electrochemical-biochemical cell-free system for the conversion of C1 substrates into the universal biological building block acetyl-CoA. The synthetic reductive formate pathway (ReForm) consists of five core enzymes catalyzing non-natural reactions that were established through a cell-free enzyme engineering platform. We demonstrate that ReForm works in a plug-and-play manner to accept diverse C1 substrates including CO2 equivalents. We anticipate that ReForm will facilitate efforts to build and improve synthetic C1 utilization pathways for a formate-based bioeconomy.

Rieske non-heme iron oxygenases (ROs) are redox enzymes that are essential for microbial biodegradation and natural product synthesis. These enzymes utilize molecular oxygen for oxygenation reactions, making them very useful in applied enzymology due to their broad reaction scope and high selectivities. The mechanism of oxygen activation in ROs involves electron transfers between redox centers of associated protein components, forming an electron transfer chain (ETC). Although the ETC is essential for electron replenishment, it carries the risk of reactive oxygen species (ROS) formation due to electron loss during oxygen activation. Our previous study linked ROS formation to O2 uncoupling in the flavin-dependent reductase of the three-component cumene dioxygenase (CDO). In the present study, we extend this finding by investigating the effects of ROS formation on the multi-component CDO system in a cell-free environment. In particular, we focus on the effects of hydrogen peroxide (H2O2) formation in the presence of a NADH cofactor regeneration system on the efficiency of CDO catalytic efficacy in vitro. Based on this, we propose the implementation of hybrid systems with alternative (non-native) redox partners for CDO, which are highly advantageous in terms of reduced H2O2 formation and increased product formation. The hybrid system consisting of the RO-reductase from phthalate dioxygenase (PDR) and CDO proved to be the most promising for the oxyfunctionalization of indene, showing a 4-fold increase in product formation (20 mM) over 24 h at a 3-fold increase in production rate compared to CDO-WT.