Biocatalysis@TUDelft

The flavoenzyme NctB from Shinella sp. HZN7 catalyzes the oxidation of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine concomitant with dioxygen reduction, which is the same chemistry catalyzed by the well-studied flavoenzyme L-6-hydroxynicotine oxidase (LHNO) from Paenarthrobacter nicotinovorans. However, while both enzymes are members of the flavoprotein amine oxidoreductase (FAO) family, they share only 26% sequence identity and are evolutionarily distant. Furthermore, nearly all FAOs (including LHNO) have a conserved lysine proximal to N5 of the flavin that is known to promote the reaction with O2 in the oxidative half-reaction, yet NctB, unusually, has a glutamate (Glu292) at this position. We report here using transient kinetics that NctB reacts rapidly with dioxygen in the oxidative half-reaction despite lacking the conserved lysine associated with promoting the reaction with O2 in FAO family enzymes. Mutagenesis reveals that a lysine derived from a different sequence position (Lys331) likely accelerates the reaction with dioxygen in NctB, as the K331M mutation results in a 1400-fold decrease in rate constant for reaction with O2. Glu292 forms a salt bridge with Lys331 in the structure of NctB, and a E292T mutation results in a [~]80-fold decrease in rate constant for reaction with O2, suggesting that Glu292 optimizes the positioning and/or properties of Lys331 to promote dioxygen activation. Analysis of pH-rate effects in NctB shows similar pH profiles as in LHNO despite having differences in active site structure. These results indicate that NctB and LHNO convergently evolved to have the same enzymatic function.

N,N-dimethylformamidase (DMFase) is a non-heme iron enzyme that catalyzes the hydrolysis of N,N-dimethylformamide (DMF) using a noncanonical Fe(III)-2Tyr-1Glu coordination motif. The precise role that this nonconventional active site plays in catalysis remains poorly understood. We performed an extensive computational investigation of DMFase catalysis, combining reaction pathway analysis with quantum mechanical cluster models, charge shift analysis, and energy decomposition analysis to identify the mechanistic role of the coordinating tyrosines/glutamate and second coordination sphere residues. We first compared two mechanisms initiated by the key second coordination sphere residues Glu657 and His519. While both mechanisms generate a ferric hydroxide intermediate, the Glu657-initiated mechanism exhibits more favorable barriers and thermodynamics. These calculations reveal distinct catalytic roles for second second-sphere residues: Glu657 facilitates direct proton transfers, His519 and Asn547 stabilize the rate-determining transition state, and Lys567 favors the anionic tyrosinate state of Tyr440 via a cation–π interaction. Mechanistic comparisons to canonical Fe(II)/Fe(III)-2His-1Glu variants reveal that coordination of Fe by tyrosine residues lowers the barrier for deprotonation of a water ligand and subsequent nucleophilic attack on DMF. Attempts to tune the active site through fluorination of coordinating tyrosinate residues yield minimal additional benefit, indicating that the native motif has finely-tuned electronic characteristics. These results demonstrate how the 2Tyr-1Glu motif and its second coordination sphere context enable hydrolytic reactivity in DMFase and suggest Glu657 and Lys567 as targets of future mutagenesis to validate their mechanistic roles.

A biocatalytic route for the enantiodivergent synthesis of cyclohexylidene-based axially chiral amines was developed. This method features imine reductase catalyzed remote stereocontrol in establishing the unusual axial chirality arising from the restricted double bond, providing both enantiomers of chiral products in high yield with high enantioselectivity.

Abstract

Cyclohexylidene-based amines exhibit unique axial chirality arising from the restricted double bond and have shown great potential in medicinal chemistry. However, their asymmetric synthesis remains challenging due to the long distance between the chirally relevant groups. Herein, we report a highly efficient and asymmetric synthesis of cyclohexylidene-based axially chiral amines from 4-substituted cyclohexanones and primary amines catalyzed by imine reductases (IREDs). Enantiodivergent IREDs were identified to provide convenient access to both enantiomers of chiral products with high yields and enantioselectivity (up to 99% yield, 99:1 or 1:99 enantiomeric ratio). A gram-scale synthesis of cyclohexylidene-based amines was also achieved. Moreover, protein X-ray crystallography and molecular modeling studies were conducted to provide structural insight into the remote stereocontrol of IREDs in generating cyclohexylidene-based axial chirality.

Furanolides represent an emerging class of natural products known for their structural diversity and potent bioactivities, including antibiotic, cytotoxic, or algicidal effects. The systematic exploration of their biological activity profiles for the discovery of new hits for drug development thus constitutes a promising endeavor. However, their low natural abundance and the resulting difficul-ty in obtaining sufficient quantities have limited further in-depth investigations into their biological activity and structure-activity relationships (SAR). Building on our recent discovery of biosynthetic enzymes catalyzing furanolide core-structure assembly, we herein developed a cost-effective, one-pot enzymatic toolbox that enables the fast generation of hundreds of furanolide structural analogs. We systematically evaluated antimicrobial activities and cytotoxicity against the A549 lung cancer cell line for a repre-sentative selection of library congeners and explored their SAR. Several derivatives demonstrated significant cytotoxicity, particu-larly against lung cancer stem cells, offering promising insights into the development of furanolides as potential anticancer agents. Additionally, some analogs displayed promising antibacterial activity against important Gram-positive pathogens such as Staphylo-coccus aureus.

The discovery of a new extremophile alga, Chlamydomonas pacifica, provides an opportunity to expand on heterologous protein expression beyond the traditional Chlamydomonas reinhardtii. C. pacifica is a unicellular extremophile capable of surviving at high pH, high temperatures, and high salinity. These various growth conditions allow C. pacifica to outcompete any invading contaminants in open-air environments. Developing this novel species as a platform for recombinant protein production could significantly advance commercial microalgal recombinant protein production. We have previously shown that C. reinhardtii can secrete a plastic-degrading enzyme: a PETase known as PHL7. This PETase is capable of cleaving ester bonds and has been used commercially for the degradation of PET plastics. However, the expression of such an enzyme has yet to be done in open raceway ponds and on a large scale. Here, we describe the culturing of PHL7 transgenic C. pacifica strain in three 80L raceway ponds and the measurements of recombinant enzymatic expression and activity found in the culture media. Our work provides proof of concept that this new organism can produce functional PHL7 enzymes in addition to producing the valuable components that inherently exist in the C. pacifica algae biomass. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/654053v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@4da5feorg.highwire.dtl.DTLVardef@1ccd743org.highwire.dtl.DTLVardef@148958forg.highwire.dtl.DTLVardef@52f36f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Scoring biomolecular complexes remains central to structural modeling efforts. Recent studies suggest that AlphaFold (AF) - a revolutionary deep learning model for biomolecular structure prediction - has implicitly learned an approximate biophysical energy function. While many researchers highly rely on AF-derived scores for structure evaluation, existing AlphaFold2-based implementations require iterative refinement of the input structure, leading to biased scoring. To address this limitation, we adapted AlphaFold3 into a score-only model, AF3Score, by directly feeding input coordinates into the confidence head while bypassing the diffusion-based structure module. AF3Score demonstrates robust performance in structural quality assessment across diverse systems including monomeric proteins, protein-protein complexes, de novo designed binders, and fold-switching proteins. In benchmarking designed binder screening, AF3Score outperformed state-of-the-art methods for 8 out of 10 targets. Moreover, combining AF3Score with AlphaFold2-derived methods significantly improved the enrichment of experimentally validated binders, increasing the success rate from 15.2% to 31.6%. Additionally, AF3Score effectively identified stable conformations in fold-switching proteins, whereas AlphaFold predominantly predicted only the dominant fold. These findings highlight the broad applicability of AF3Score, from high-throughput screening in de novo binder design to filtering docking-generated poses and molecular dynamics (MD) trajectories.

Base editing allows for the precise modification of genetic information, providing new avenues for treating diseases1. The adenine base editor ABE8e is currently the most efficient and widely used tool for adenine base editing. ABE8e was developed through multiple rounds of directed evolution of Escherichia coli tRNA adenine deaminase, including phage-assisted continuous evolution (PACE)2,3. While PACE is highly effective, it is a complex system that poses challenges for implementation4. Despite its high efficiency, ABE8e is associated with limitations such as relatively higher bystander editing effects and elevated off-target activity5, which need to be addressed to further enhance its precision and safety. Here, we developed a novel method for the de novo discovery of evolved ABE components, particularly adenine deaminases. This process involves identifying candidate proteins through AI-based structural prediction and clustering, followed by the enhancement of deaminase editing activity through screening libraries created by sequential amino acid saturation mutagenesis. This evolutionary strategy simplifies the approach by employing saturation mutagenesis libraries tailored to specific segments, thereby enabling exploration of an expanded sequence space and increasing the likelihood of discovering adenine deaminases with superior capabilities. The newly developed hpABE5.20 here demonstrates a more refined editing window, reduced DNA off-target effects that are both sgRNA-dependent and -independent, and minimized RNA off-target activity, while maintaining robust editing efficiency relative to ABE8e. Furthermore, hpABE5.20 has been successfully applied for precise and effective therapeutic adenine base editing in cellular disease models, humanized mice, and non-human primates.

In mycobacteria, the protein EtfD is thought to link {beta}-oxidation of fatty acids with the electron transport chain, two processes that have attracted attention as targets for therapeutics to treat tuberculosis (TB) and other mycobacterial infections. It has been proposed that targeting {beta}- oxidation could shorten treatment duration by killing non-replicating Mycobacterium tuberculosis within granulomas in the lungs. Here we show that Mycobacterium smegmatis, a fast growing and nonpathogenic model for energy metabolism in M. tuberculosis, relies on EtfD for extracting energy from {beta}-oxidation. Electron cryomicroscopy allowed structure determination of M. smegmatis EtfD, revealing an unusual linear [3Fe-4S] cluster that has not been seen in other protein structures, but which resembles the catalytic noncubane [4Fe-4S] clusters in heterodisulfide reductases. The structure suggests how EtfD transfers electrons from {beta}-oxidation to the electron transport chain. We devised an assay that couples EtfD activity to a fluorescent readout of proton pumping by the electron transport chain, which can be used to identify compounds that block mycobacteria from using {beta}-oxidation to power oxidative phosphorylation.

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.

Ribosomes synthesize proteins with an RNA-only active site across all kingdoms of life. Yet, despite decades of high-resolution ribosome structures, the precise catalytic mechanism has remained elusive. Here, we provide structural evidence for metal ion involvement in peptide bond formation, drawn from ribosome structures spanning bacteria, archaea, and eukaryotes. The metal ion resides in the active site, adjacent to a universally conserved stack of three base triples, reminiscent of the catalytic triplex in group II introns and the spliceosome, which catalyze pre-mRNA splicing. Metal-ion catalysis thus emerges as a fundamental unifying theme across the central dogma spanning protein synthesis, RNA splicing, and nucleic acid replication.

Alpha-synuclein (S) and tau play important roles in the pathology of Parkinsons Disease and Alzheimers Disease, respectively, as well as numerous other neurodegenerative diseases. Both proteins are classified as intrinsically disordered proteins (IDPs), as they have no stable structure that underlies their function in healthy tissue, and both proteins are prone to aggregation in disease states. There is substantial interest in understanding the roles that post-translational modifications (PTMs) play in regulating the structural dynamics and function of S and tau monomers, as well as their propensity to aggregate. While there have been many valuable insights into site-specific effects of PTMs garnered through chemical synthesis and semi-synthesis, these techniques are often outside of the expertise of biochemistry and biophysics laboratories wishing to study S and tau. Therefore, we have assembled a primer on genetic code expansion and enzymatic modification approaches to installing PTMs into S and tau site-specifically, including isotopic labeling for NMR and fluorescent labeling for biophysics and microscopy experiments. These methods should be enabling for those wishing to study authentic PTMs in S or tau as well as the broader field of IDPs and aggregating proteins.

A critical step in the global nitrogen cycle is the conversion of dinitrogen into biologically accessible ammonia. In Nature this is accomplished by the nitrogenase (N2ase) family of enzymes. Carbon monoxide (CO) has long been known as an inhibitor of dinitrogen reduction by N2ase, but it can also be a substrate of the enzyme, when it is catalytically reduced to hydrocarbons. Understanding the CO interactions with N2ases are thus relevant to both dinitrogen fixation and Fischer-Tropsch-like chemistry. Here, the interaction of CO with the a-V70I variant of Azotobacter vinelandii MoFe N2ase was investigated using EPR- and IR- monitored photolysis of bound CO under cryogenic conditions. This was supplemented by further analysis of stopped-flow FT-IR (SF-FT-IR) data under turnover conditions. The a-V70I variant adds a single methyl group close to the active site, and the results show that this inhibits and slows, but does not substantially chemically change, the binding of CO to the FeMo-cofactor. The EPR spectra of both the hi-CO and lo-CO states closely resemble those from the wild-type enzyme. Similarly, the SF-FT-IR spectrum is strikingly similar, with only small shifts in band energies and comparison with data from wild-type enzyme allows better interpretation of the published SF-FT-IR spectra. The extra carbon does, however, impact and inhibit the photochemical release and migration of CO at cryogenic temperature, resulting in novel CO-bound species. In particular, a photolysis product species, termed Hi-1*, with an IR band at 1944 cm-1 may involve CO photochemically migrating on the FeMo-cofactor.

Conventional approaches to heterologous gene expression rely on codon optimization, which is limited to swapping synonymous codons and often fails to capture deeper adaptive changes. In contrast, naturally evolved orthologous genes between species often differ by more than just synonymous substitutions - they can include non-synonymous mutations, insertions, and deletions that confer functional adaptation to different host contexts. Here we present OrthologTransformer, a Transformer-based deep learning model that converts orthologous genes between species by learning from large-scale orthologous gene datasets curated for high-quality sequence alignments. The model recapitulates the full spectrum of evolutionary differences - from synonymous codon swaps to amino acid-changing mutations and indels - to predict a coding sequence optimized for a target species while preserving the protein's function. In extensive validation across diverse bacterial species pairs, OrthologTransformer significantly increased the conversion accuracy of generated genes to native target sequences compared to the original source genes, even for pairs with stark disparities in GC content and optimal growth temperature. The Transformer's context-aware designs also favored conservative amino acid substitutions, maintaining protein functional integrity. As a proof-of-concept, an OrthologTransformer-designed PETase gene for Bacillus subtilis from the host species Ideonella sakaiensis was synthesized and expressed, yielding robust PET plastic-degradation activity that surpassed synonymous codon-optimized controls. These results establish OrthologTransformer as a powerful tool for de novo cross-species gene adaptation, transcending the limits of traditional codon optimization and enabling more effective heterologous gene performance in synthetic biology applications.

Nature Chemistry, Published online: 14 May 2025; doi:10.1038/s41557-025-01822-y

Some thiamine diphosphate-dependent enzymes are powerful biocatalysts, forming carbon–carbon bonds between two α-keto acids, but their catalytic properties have been poorly understood. Now the substrate selectivity and catalytic stereoselectivity of two representative enzymes, CsmA and BbmA, have been fully characterized. Enzymatic total synthesis using these enzymes generates diverse carboligation products.

Instead of the N-nitration, oxidation of hydrazine and hydrazone is involved in N-nitroglycine biosynthesis to form the nitramine structure. In vivo and in vitro results indicated the N-nitroglycine biosynthetic pathway involves serine incorporation induced by NRPS, and a nonheme diiron N-oxygenase and a cytochrome P450s as the pivotal oxidative enzymes responsible for nitramine formation.

Abstract

N-nitroglycine (NNG), a rare nitramine natural compound, is the only known example produced by streptomyces strains. In this study, we clarified that NNG biosynthesis originates from glycine and l-lysine and elucidated its biosynthetic gene cluster (BGC) nng. This cluster shares high homology with the recently reported BGCs of diazo compound azaserine. In vivo and in vitro results have indicated NNG biosynthetic pathway involves hydrazine and hydrazone generation, with a non-heme diiron N-oxygenase and a cytochrome P450 responsible for forming the nitramine structure. Furthermore, although NNG lacks a serine unit in the structure, its biosynthesis still requires the incorporation of a serine attached to the hydrazone intermediate by non-ribosomal peptide synthetase (NRPS), similar to the azaserine pathway, and two hydrolases are putatively involved in thioester hydrolysis and serine removal, respectively. This study, along with comparisons to azaserine biosynthesis, not only paves the way for the discovery of new nitramine and diazo compounds by genome mining but also highlights the potential for uncovering novel enzymes and chemistry involved in hydrazone oxidation.

Through the directed evolution of an underexploited nonheme Fe extradiol dioxygenase, we developed a unified cooperative photobiocatalytic strategy to allow for three types of enantioconvergent radical transformations, including azidation, thiocyanation, and isocyanation. Computational studies based on density functional theory (DFT) and molecular dynamics (MD) simulations suggested a π-facial selective radical rebound mechanism as the enantiodeter

Abstract

Cooperative catalysis with an enzyme and a small-molecule photocatalyst has recently emerged as a potentially general activation mode to advance novel biocatalytic reactions with synthetic utility. Herein, we report cooperative photobiocatalysis involving an engineered nonheme Fe enzyme and a tailored photoredox catalyst to achieve enantioconvergent decarboxylative azidation, thiocyanation, and isocyanation of redox-active esters via a radical mechanism. We repurposed and further evolved metapyrocatechase (MPC), a nonheme Fe extradiol dioxygenase not previously studied in new-to-nature biocatalysis, for the enantioselective C─N₃, C─SCN, and C─NCO bond formation via an enzymatic Fe─X intermediate (X═N₃, NCS, and NCO). A range of primary, secondary, and tertiary alkyl radical precursors were effectively converted by our engineered MPC, allowing the syntheses of organic azides, thiocyanates, and isocyanates with good to excellent enantiocontrol. Further derivatization of these products furnished valuable compounds including enantioenriched amines, triazoles, ureas, and SCF₃-containing products. DFT and MD simulations shed light on the mechanism as well as the binding poses of the alkyl radical intermediate in the enzyme active site and the π-facial selectivity in the enantiodetermining radical rebound. Overall, cooperative photometallobiocatalysis with nonheme Fe enzymes provides a means to develop challenging asymmetric radical transformations eluding small-molecule catalysis.