ChemBioChem

In their recent communication in Angewandte Chemie (10.1002/anie.202508565), Suk Min Kim and coworkers have described the effect of modifying the gas channels of the CO dehydrogenase II from Carboxydothermus hydrogenoformans, an enzyme that oxidizes reversibly CO into CO2. Their goal was to use mutagenesis to slow down the arrival of O2 at the active site. They reported a large increase in the resistance against oxygen, one of the major barriers to the application of this extremely fast and efficient enzyme in biotechnological devices, with an increase in the IC50 of more than two orders of magnitudes for some variants, with only a minor impact on the affinity of the enzyme for CO. We have produced the same variants, and characterized them in depth using Protein Film Electrochemistry. We used an approach that has proven very useful to learn and understand about the reactivity of CO dehydrogenases (and other redox enzymes like hydrogenases) with O2. We found that, contrary to the claims by Kim and coworkers, the A559W and the A559W/V610H mutants are not more resistant than the WT against oxygen.

Synthetic reactions often require solvents incompatible with biocatalysts. Here, we encapsulate a de novo heme-containing enzyme, C45, in calcium-alginate hydrogel beads to facilitate heterogeneous biocatalysis in neat organic solvents. Post-encapsulation, C45 retains activity even when the beads are suspended in organic solvents. In particular, the carbene transferase activity of C45 is enhanced when reactions are performed in aprotic, non-polar solvents such as hexane and toluene. Activity-solvent dependencies reveal that this activity boost is likely due to beneficial partitioning of the substrate into the beads from the organic phase. Furthermore, encapsulation facilitates enzyme recovery and recycling after the reaction. Such encapsulation opens up novel opportunities for biocatalysis in organic solvent systems, combining desired solvent properties of organic chemistry with enzymatic selectivity and proficiency.

Polyketides constitute a large class of natural products with important biological activities and applications such as antibiotics, antitumor agents, pesticides, and pigments. Their biosynthesis is catalyzed by polyketide synthases (PKSs) which are multi-domain enzymes evolutionarily related to fatty acid synthases (FASs). Despite their close homology in structure and the chemistry they perform, FASs and PKSs differ fundamentally in their catalytic programming: FASs run fully reducing elongation reactions to yield saturated fatty acids, while iterative PKSs execute reductions just in selected cycles, generating complex oxidized compounds. In this study, we aimed at engineering the metazoan FAS in its KR domain to switch from fully reducing to a non-reducing mode during chain elongation. Guided by recent insights into KR programming, we incorporated a helix into metazoan FAS, which is found in KRs from iterative PKSs and type II FASs with chain length programming. These FAS variants initially catalyze complete fatty acid cycles but lose the ability of {beta}-keto reduction in later elongation rounds, producing intermediates that spontaneously cyclize to pyrone products. Finally, our study provides valuable insight into the mechanism of KR catalysis identifying another amino acid next to the active tyrosine which is capable for intermediate protonation.

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.

Rieske oxygenases (ROs) are enzyme systems involved in microbial biodegradation or late-stage modifications during natural product biosynthesis. A major obstacle to working with ROs is their dependence on multi-component electron transfer chains (ETCs). Thereby, electrons from NAD(P)H are shuttled directly via a reductase (Red) or indirectly via an additional ferredoxin (Fd) to a terminal oxygenase (Oxy) for oxygen activation and subsequent substrate conversion. The present work evaluates potential fusion strategies to simplify the ETC of the three-component cumene dioxygenase (CDO) from Pseudomonas fluorescence. In in vitro reactions, the fusion of CDO-Red to CDO-Fd is the most suitable for activation of CDO-Oxy with product formation of approximately 22 mM (72 % conversion). Furthermore, protein fusion to CDO-Oxy was found to be feasible, highlighting the versatility of the redox partner fusion approach. Overall, this study aims to contribute to the research field of ROs by providing a promising strategy to simplify their multi-component nature.

BackgroundBiotechnological applications are steadily growing and have become an important tool to reinvent the synthesis of chemicals and pharmaceuticals for lower dependence on fossil resources. In order to sustain this progression, new feedstocks for biotechnological hosts have to be explored. One-carbon (C1-)compounds, including formate, derived from CO2 or organic waste are accessible in large quantities with renewable energy, making them promising candidates. Previous studies showed that introduction of the formate assimilation machinery from Methylorubrum extorquens into Escherichia coli allows assimilation of formate into the established biotechnological host. Applying this established route for formate assimilation, we here investigated utilisation of formate for the production of value-added building blocks in E. coli using S-adenosylmethionine (SAM)-dependent methyltransferases. ResultsWe first analysed methylation activity in E. coli BL21 with a two-vector system to produce three different methyltransferases together with the formate assimilation machinery. Feeding isotopically labelled formate, products with 51 - 81% 13C-labelling could be obtained by maintaining in vivo methylation activity. Focussing on improvement of in vivo methylation, we analysed two further E. coli strains with an engineered C1-metabolism and, following condition optimisation, achieved a doubled methylation activity with a share of more than 70% formate-derived methyl groups. ConclusionsThis study demonstrates the efficient transformation of formate into methyl groups in E. coli. Our findings support that feeding formate can improve the availability of usable C1-compounds and, as a result, increase in vivo methylation activity in engineered E. coli.

Fluorinated amino acids are a promising entry point for incorporating new-to-Nature chemistries in biological systems. Hence, novel methods are needed for the selective synthesis of these building blocks. In this study, we focused on the enzymatic synthesis of fluorinated alanine enantiomers. To this end, the alanine dehydrogenase from Vibrio proteolyticus and the diaminopimelate dehydrogenase from Symbiobacterium thermophilum were applied to the in vitro production of (R)-3-fluoroalanine and (S)-3-fluoroalanine, respectively, using 3-fluoropyruvate as the substrate. Additionally, an alanine racemase from Streptomyces lavendulae, originally selected for setting an alternative enzymatic cascade leading to the production of these non-canonical amino acids, had an unprecedented catalytic efficiency in the {beta}-elimination of fluorine from the monosubstituted fluoroalanine. The in vitro enzymatic cascade based on the dehydrogenases of V. proteolyticus and S. thermophilum included a cofactor recycling system, whereby a formate dehydrogenase from Pseudomonas sp. 101 (either native or engineered) coupled formate oxidation to NAD(P)H formation. Under these conditions, the reaction yields for (R)-3-fluoroalanine and (S)-3-fluoroalanine reached >85% on the fluorinated substrate and proceeded with complete enantiomeric excess. Moreover, the selected dehydrogenases were also able to catalyze the conversion of trifluoropyruvate into trifluorinated alanine, as a first-case example of biocatalysis with amino acids carrying a trifluoromethyl group.

Multinuclear nonheme iron-dependent oxidases (MNIOs) constitute one of the largest families of enzymes involved in natural product biosynthesis. Distinct MNIO subfamilies utilize molecular oxygen to catalyze a wide variety of complex peptide rearrangements, including {beta}-carbon excision and heterocyclization. Highly homologous MNIOs have been proposed to install either oxazolone-thioamides or 5-thiooxazoles as cysteine post-translational modifications in the closely related bufferin and EGKCG families of peptide chalkophores. These alternative structures prompted discussion of the subtle mechanistic features of MNIO enzymes that might determine reaction outcome. Here, we combine uniform 15N labeling with cysteine-specific carbonyl 13C labeling to unambiguously assign 5-thiooxazoles as the cysteine modifications in bufferins. Together with the recent identification of 5-thiooxazoles in three members of the sister EGKCG family and the re-assignment of the cysteine modification in oxazolin, these findings confirm that closely related MNIOs catalyze identical post-translational modifications.

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.

Molecular editing of an amino group from -position of amino acids to its {beta}-position is of scientific interest and could be economically appealing. Here we reconstructed an in vitro biotransformation pathway composed of two cascade decarboxylases, i.e., aspartate {beta}-decarboxylase and aspartate -decarboxylase, and implemented molecular editing to change -alanine into {beta}-alanine. In it, we discovered a new reaction of aspartate {beta}-decarboxylase that can fix CO2 directly. This cascade enzymatic pathway enabled an aminomutation reaction with 100% carbon atom economy. This work presented the first CO2-fixing biological reaction catalyzed by the amino acid decarboxylases and demonstrated a new means for the molecular editing of -amino acids.

Phosphorylation is a highly regulated protein post-translational modification catalyzed by kinases. Kinases and phosphorylated proteins are key players in a myriad of cellular events, including cell signaling. When cell signaling networks are improperly regulated by kinases, various pathologies can arise, such as cancers and neurodegenerative disease. With critical roles in normal and disease biology, kinase-substrate interactions must be thoroughly characterized. Previously, the chemoproteomic method, kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP), was developed to identify the kinases of a phosphoprotein substrate of interest. Here, K-CLIP was modified to profile the substrates of a kinase of interest. Specifically, the substrate profile of cAMP-dependent protein kinase (PKA) was studied with K-CLIP using a new ATP analog, ATP-alkyne aryl azide. Kinase-focused K-CLIP discovered SMC3 as a PKA substrate. With versatility for any kinase or phosphoprotein substrate of interest, K-CLIP will expand our understanding of kinase-mediated cell biology in healthy and diseased states.

Nar1 is an essential eukaryotic protein proposed to function as an iron-sulfur (Fe/S) cluster trafficking factor in the cytosolic iron-sulfur assembly (CIA) machinery. However, such a role has remained unclear due to difficulties in purifying adequate amounts of cofactor-bound protein. The [FeFe]-hydrogenase-like protein has two conserved binding sites for [4Fe-4S] clusters, one of which is predicted to be a labile site for cluster transfer to downstream targets. Here, we report a new preparation procedure for Nar1 that facilitated studies by UV-Vis, EPR, and Mossbauer spectroscopies, along with native mass spectrometry. Nar1 recombinantly produced in E. coli contained a [4Fe-4S] cluster, bound presumably at site 1, along with an unexpected [2Fe-2S] cluster bound at an unknown site. Fe/S reconstitution reactions installed a second [4Fe-4S] cluster at site 2, leading to protein with three Fe/S cofactors. Strikingly, one [4Fe-4S] cluster was rapidly destroyed by molecular oxygen, potentially linking Nar1 oxygen sensitivity to phenotypes observed previously in vivo. These advances now allow for the pursuit of in vitro Fe/S cluster transfer assays, which will shed light on Fe/S trafficking by CIA components and how they may facilitate the insertion of [4Fe-4S] and potentially [2Fe-2S] clusters into target proteins in the cytosol.

Cyanide (CN) toxicity is traditionally understood to result from its binding of hemeFe centers, thereby disrupting mitochondrial cytochrome oxidase function and oxygen utilization by other globin proteins. Recently, a diffusible reactive oxygen species (DROS) mediated reaction mechanism called murburn concept was proposed to explain mitochondrial ATP-synthesis and heat generation. Per this purview, it was theorized that CN ion-radical equilibrium dissipates the catalytically vital DROS into futile cycles, producing water. In the current study, a comparative quantitative assessment of the above two explanations is made for: (i) lethal dosage or concentrations of CN, (ii) thermodynamics and kinetics of the binding/reaction, and (iii) correlation of CN with the binding data and reaction chemistry of H2S/CO. The quantitative findings suggest that the hemeFe binding-based toxicity explanation is untenable. CN also inhibited the experimental in vitro DROS-mediated coupling of inorganic phosphate with ADP. Further, pH-dependent inhibition profiles of heme enzyme catalyzed oxidation of a phenolic (wherein an -OH group reacts with DROS to form water, quite akin to the murburn model of ATP synthesis) indicated that- (i) multiple competitive reactions in milieu controlled outcomes and (ii) low concentrations of CN cannot disrupt activity via a coordination (binding) of cyanide at the distal hemeFe. Therefore, the M-level IC50 and the acutely lethal effect of CN on cellular respiration could be explained by the deleterious interaction of CN ion-radical equilibrium with DROS in matrix, disrupting mitochondrial ATP synthesis. This work supports the murburn explanation for cellular respiration.

Site-specific attachment of biorthogonal handles to proteins is an essential tool in chemical biology research and diverse applications including imaging and protein immobilization, as well as for the development of next-generation therapeutics such as antibody drug-conjugates. Among the available methods, enzymatic post-translational modification of short protein tags offers precision, stability, and modularity. However, broader application is often limited by complex substrate syntheses, the requirement of long or rigid recognition tags, and limited reaction efficiencies. Here, we present ADDing, a straightforward enzymatic method for functionalizing proteins with click chemistry handles using the flavin transferase ApbE. We discovered that, given a dedicated adenine diphosphate derivative (ADD) substrate, the enzyme attaches a phosphoribosyl moiety bearing bioorthogonal handles to proteins featuring a DxxxGAT amino acid motif. As the substrates can easily be enzymatically synthesized from NAD and inexpensive precursors, ADDing click handles can be performed in a streamlined, one-pot workflow combining substrate synthesis and protein conjugation. ADDing allows rapid, high-yield functionalization of proteins featuring the recognition tag at either terminus or internal loops and is compatible with copper and copper free azide-alkyne cycloaddition reactions. To demonstrate its broad applicability, we performed a wide variety of protein functionalizations, including fluorescent labeling, protein-protein, protein-DNA conjugation, and protein immobilization. This versatile technology thus holds great potential for chemical biology and the production of biological therapeutics.

Tuberculosis (TB) is an infectious disease caused mainly by Mycobacterium tuberculosis (Mtb) and is responsible for millions of deaths. New Mtb strains resistant to TB drugs are emerging and spreading. The first-line TB drug, isoniazid (INH), must be activated inside mycobacterial cells by the catalase-peroxidase enzyme KatG to exert its antimicrobial activity, and mutations on the katG gene are a significant cause of INH resistance in clinics. The metal-containing compound IQG-607 is an INH analog developed to inhibit the target of INH, the FASII enzyme enoyl-ACP-reductase (InhA), without requiring KatG. However, we recently showed that inside mycobacterial cells, IQG-607 activity depends on KatG. Hence, this compound might also be activated by KatG to inhibit InhA. We evaluated whether recombinant MtKatG uses IQG-607 as a substrate in oxidation reactions and adduct formation with NAD+. A recombinant MtKatG was produced in E. coli and purified in a 3-step protocol to obtain a homogeneous protein. An HPLC method was optimized to monitor both oxidation and adduct products, and our assay system was validated by performing control reactions using INH as a substrate. We found that the metal-based compound IQG-607 is not a substrate for recombinant MtKatG under all conditions tested.

A modified protein fragment complementation assay has been designed and validated as a gain-of-signal biosensor for nucleic acid:nucleic acid interactions. The assay uses fragments of NanoBiT, the split luciferase reporter enzyme, that are esterified at their C-termini to steramers, sterol-modified oligodeoxynucleotides. The Drosophila hedgehog autoprocessing domain, DHhC, served as a self-cleaving catalyst for these bioconjugations. In the presence of ssDNA or RNA with segments complementary to the steramers and adjacent to one another, the two NanoBiT fragments productively associate, reconstituting NanoBiT enzyme activity. NanoBiT luminescence in samples containing nM ssDNA or RNA template exceeded background by 30-fold and as high as 120-fold depending on assay conditions. A unique feature of this detection system is the absence of a self-labeling domain in the NanoBiT bioconjugates. Eliminating that extraneous bulk broadens the detection range from short oligos to full-length mRNA. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/572427v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@f0706dorg.highwire.dtl.DTLVardef@1653d01org.highwire.dtl.DTLVardef@18843bcorg.highwire.dtl.DTLVardef@1170e47_HPS_FORMAT_FIGEXP M_FIG C_FIG

Single molecule mass photometry was used to study the dynamic equilibria of the ubiquitous and highly abundant 2-Cysteine peroxiredoxins (2-CysPRX). 2-CysPRXs adopt distinct functions in all cells dependent on their oligomeric conformation ranging from dimers to decamers and high molecular weight aggregates (HMW). The oligomeric state depends on the redox state of their catalytic cysteinyl residues. To which degree they interconvert, how the interconversion is regulated, and how the oligomerisation propensity is organism specific remains, however, poorly understood. The dynamics differs between wild-type and single point mutants affecting the oligomerization interfaces, with concomitant changes to function. Titrating concentration and redox state of Arabidopsis thaliana and human 2-CysPRXs revealed features conserved among all 2-CysPRX and clear differences concerning oligomer transitions, the occurrence of transition states and the formation of HMW which are associated with chaperone activity or storage. The results indicate functional differentiation of human 2-CysPRXs. Our results point to a diversified functionality of oligomerization for 2-CysPRXs and illustrate the power of mass photometry to non-invasively quantify oligomer distributions in a redox environment. This knowledge is important to fully address and model PRX function in cell redox signaling e.g., in photosynthesis, cardiovascular and neurological diseases or carcinogenesis.

The malarial parasite Plasmodium falciparum cleaves host hemoglobin by cascade of proteolytic enzymes. The cysteine protease, Falcipain-2 (FP2) plays an essential role in the process and important for parasite survival, making it a potential drug target. However, similarities with host cysteine cathepsins hamper selective inhibition, thus necessitates detailed structural and functional characterizations of FP2. The present study uncovers a novel regulatory role of polyethylene glycol 400 (PEG400) on FP2 activity. PEG400 inhibits FP2 activity on small peptide substrate and azo-casein, while enhancing hemoglobin degradation, rendering a dual effect on FP2 catalysis. A mixed-type of inhibition has been observed for PEG400 against small peptide substrate of FP2, consistent with binding of PEG400 to catalytic cleft, confirmed by fluorescence quenching and docking studies. Unlike typical nonspecific PEG-protein interactions, PEG400 adopts a fit within catalytic region of FP2 and partially overlaps with leupeptin binding sites, albeit with lower affinity. Computational analysis further identifies a novel allosteric binding pocket of PEG400, supported by in-silico mutagenesis and molecular dynamics simulation. This pocket exhibits minimal conservation in human cathepsins, suggesting selective potential. In contrast to this inhibitory role, biochemical assay reveals that PEG400 promotes haemoglobin proteolysis. Spectroscopic analyse suggests PEG400 alter hemoglobin structural dynamics to favour proteolysis. ENM based normal mode analysis reveals upon haemoglobin binding, PEG400 restricts FP2 hinge-bending motion, improves FP2-hemoglobin proximity, and simultaneously PEG400 is dislodged from the active site, thereby promoting proteolysis. The combined experimental and computational findings reveal a novel mechanism of FP2 regulation, opening new therapeutic avenues.

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.

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.