Biocatalysis@TUDelft

A biocatalytic strategy was developed for the synthesis of enantioenriched halogenated cyclopropanes via enzyme-catalyzed cyclopropanation of chloro- and bromo-substituted olefins in the presence of diazoacetonitrile with engineered myoglobins. This method was applied to the transformation of a range of alpha- and beta-chloro/bromo vinylarenes with good to excellent diastereo- and enantioselectivity (up to 99% de and ee).

Abstract

Halogenated cyclopropanes are highly desirable building blocks for both agrochemical and medicinal chemistry due to their unique structural features and ability to interact with biological targets through halogen bonds. However, methods for the highly diastereo- and enantioselective synthesis of these molecules are underdeveloped. Herein, we report the development of a new biocatalytic strategy to efficiently synthesize enantioenriched halogenated cyclopropanes through the enzymatic cyclopropanation of chloro- and bromo-substituted olefins in the presence of diazoacetonitrile using engineered myoglobin-based catalysts. This method tolerates a wide range of chloro- and bromo-containing olefins at both α- and β-positions with high yields (up to 75%) and excellent diastereo- and enantioselectivity (up to 99% de and ee). These transformations provide access to enantioenriched halogenated cyclopropanes that are challenging to prepare using existing methods, highlighting the value of engineered carbene transfer biocatalysts toward the asymmetric synthesis of enantiopure halogenated cyclopropane building blocks for applications in medicinal chemistry, agrochemicals, and target-directed synthesis.

Non-canonical redox cofactors (NRCs) are promising alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) for biomanufacturing due to low cost and exquisite electron delivery control, yet their adoption is limited by the scarcity of compatible enzymes. Here, we screened the aldehyde dehydrogenase (ALDH) protein family and identified a conserved RH/QxxR sequence motif that enables widespread NRC activity among natural enzymes. Bos taurus ALDH3a1 and Pseudanabaena biceps ALDH exhibit unprecedented turnover with nicotinamide mononucleotide (NMN+), with kcat values matching or exceeding that of NAD+ and surpassing most engineered NRC-active enzymes by 10 to 105-fold, based on the relative NRC to native activity. Structural and dynamic analyses reveal this motif reinforces cofactor positioning and pre-organizes the active site without dependence on the adenosine monophosphate moiety of NAD+. When introduced into diverse ALDH scaffolds, the RH/QxxR motif enhances NMN+ activity up to 60-fold. In addition to NMN+, this motif also supports activity across multiple non-nucleotide, simple synthetic NRCs such as 1-(2-carbamoylmethyl)nicotinamide (AmNA+). These findings elucidate Natures solution to the engineering challenge of obtaining NRC-active enzymes and offers a blueprint to mine latent evolutionary plasticity in natural enzymes that serve as superior engineering starting points.

Nature Catalysis, Published online: 01 August 2025; doi:10.1038/s41929-025-01388-5

The mechanism by which the [NiFe4S4] cluster of carbon monoxide dehydrogenases (CODHs) catalyses CO2 reduction is poorly understood. Now the structures of all catalytically relevant states of a CODH are solved, revealing the dynamics of the cluster during turnover and the role of Ni in CO2 activation.

Nature Chemistry, Published online: 25 July 2025; doi:10.1038/s41557-025-01872-2

Current tools for protein bioconjugation at the C-terminal have limited yields. Now, an enzymatic strategy for ATP-dependent activation of protein and peptide C termini has been developed. This versatile tool can be deployed for synthesis of C-terminal thioesters that enhance the yield and accessibility of diverse protein bioconjugation methods.

Nature Chemistry, Published online: 01 August 2025; doi:10.1038/s41557-025-01883-z

While intellectual virtues might be more within the remit of philosophers, many scientists would also see them as important within their domain. Dominic T. Chaloner, Michelle Francl, and T. Ryan Byerly consider what it takes to train up virtuous chemists

Enzyme-mediated divergent reactions are yielding an identical product, symbolizing “All roads lead to Rome”. In their Research Article (e202506614), Shengying Li and co-workers highlight novel degradation routes of carboxyl-free aromatic environmental pollutants mediated by CYP152 peroxygenases, using H₂O and H₂O₂ to converge structurally diverse substrates into a single catechol product via two distinct mechanisms, exemplifying enzymatic versatility.

Natural alcohol and sugar dehydrogenases that utilize the redox cofactor pyrroloquinoline quinone (PQQ) can be repurposed for enantioselective photoredox catalysis. Upon blue-light irradiation, redox-neutral radical cyclizations are catalyzed. This work adds a new class of enzymes to the toolbox of photobiocatalysis.

Abstract

Photoenzymatic catalysis facilitates stereoselective new-to-nature chemistry under mild conditions. In addition to the rational design of artificial photoenzymes, naturally occurring redox enzymes can be repurposed to promote photoredox catalysis in the chiral protein environment. Here, we show that enzymes utilizing the pyrroloquinoline quinone (PQQ) cofactor expand the toolbox of photobiocatalysis. PQQ absorbs visible light and is capable of single-electron transfer. It thus exhibits mechanistic similarities to flavin cofactors, which are widely used for photoenzymatic approaches. First, we established the trimethyl ester PQQMe₃ as a stand-alone photoredox catalyst in pure organic solvent. Upon excitation, PQQMe₃ enables the redox-neutral radical cyclization of an N-(bromoalkyl)-substituted indole. We then tested a panel of PQQ-dependent sugar and alcohol dehydrogenases for photoenzymatic catalysis in aqueous buffer, focusing on a redox-neutral radical reaction to form oxindoles. Under optimized reaction conditions, we obtained a 69% yield and an 82:18 enantiomeric ratio. Our work thus demonstrates that PQQ enzymes are capable of stereoselective photoredox catalysis. Future enzyme engineering efforts based on computational modeling and directed evolution will fully unlock their synthetic potential.

The structure of the (S)-selective “tyrosine” imine reductase (IRED) IR91 from Kribbella flavida is presented in complex with its ketone substrate and (S)-amine product, revealing for the first time the interactions of substrate and product with an IRED of this type acting in a reductive aminase mode.

Imine reductases with an (S)-preference for the reduction of the model substrate 2-methyl pyrroline typically contain tyrosine in the active site (Y-IREDs) instead of the aspartate present within (R)-selective enzymes (D-IREDs). As with D-IREDs, a subset of Y-IREDs is capable of enabling reductive amination reactions between some ketone and amine partners to give optically active amines with high optical purity. However, structures of Y-IREDs in complex with the substrates and products of the reductive amination have not been forthcoming. Herein, structures of the Y-IRED IR91 from Kribbella flavida in complex with 5-methoxy-2-tetralone, a synthetic precursor to the anti-Parkinson's treatment rotigotine, and also its reductive amination product with methylamine, 5-methoxy-(S)-2-(N-methylamino)-tetralin, are presented. The structures, in combination with mutation and kinetic studies, support a role for tryptophan residue W258 in the activity of the enzyme, possibly in binding of the ketone prior to reaction with methylamine.

Background: Enantiopure drugs offer better specificity, higher therapeutic index, and optimized pharmacokinetics compared to racemic drugs. However, pharmaceutical companies continue manufacturing racemic medicines, among others, due to the lack of tools for in situ determination of the optical purity and the absolute configura-tion, which is necessary for approval of drug substance by the European Medicines Agency (EMA) or the US Food and Drug Administration (FDA). Currently used methods for the absolute configuration and enantiomeric excess determination have significant limitations (require single crystal, UV-active chromophores, chiral reagents, etc). Results: Addressing this need of pharmacological industry for the effective analysis of chiral drugs in situ in solu-tion, we have designed and constructed the at-line setup based on Raman Optical Activity (ROA) and equipped with the 3D-printed flow reactor dedicated for fast in situ and in-flow analysis of chiral compounds. We have validated our approach by the determination of the enantiomeric excess (EE) of pure and multi-component chiral samples showing that the developed solution combines the excellent stereosensitivity, possibility to measure chi-ral samples in-flow, and a rather unique capability of direct discrimination of components of chiral mixtures. The quantitative analysis based on Partial Least Squares (PLS) regression has demonstrated that the accuracy and pre-cision of the method is very high with the average prediction error from cross-validation (RMSECV) not exceeding 1.35% (for R2 at least 99.95) for ROA spectra recorded in 20 minutes. Significance: According to our best knowledge, our work is a first example of successful in-flow and in situ ROA evaluation of chiral purity in chiral mixtures. Our direct approach does not require sample preparation, preliminary separation of enantiomers or derivatization. In the long-term, our solution paves the way toward operando (in-line) ROA, enabling real-time monitoring of chirality in reaction mixtures.

Advances in organic and gas waste valorization have enabled high-yield production of carboxylic acids, positioning them as promising feedstocks for the bioeconomy. However, carboxylic acids must be activated before downstream use, typically requiring ATP, CoA, or reduced ferredoxin to overcome unfavorable thermodynamics. These activators are costly to generate and divert carboxylic acids into CO2-releasing pathways, reducing carbon efficiency. Here, we demonstrate that aldehyde dehydrogenases (ALDHs) can directly reduce carboxylic acids to aldehydes without prior activation, a process previously thought to be biologically inaccessible. Screening 133 ALDHs revealed that this activity is remarkably widespread within the protein family, enabling production of aliphatic aldehydes and alcohols, diols, and aromatic alcohols, at titers >1 g/L, in some cases, after optimization of thermodynamic driving forces. Additionally, we applied this system to upgrade waste-derived carboxylic acid effluent streams from wastewater sludge, food waste, and waste gas (CO2). This activation-free process, termed "reverse aldehyde oxidation" (rAOX), establishes a broadly applicable, energy-efficient platform for carboxylic acid valorization at 100% carbon yield. Analogous to the reverse tricarboxylic acid cycle (rTCA) and reverse {beta}-oxidation (rBOX), rAOX exemplifies that metabolic reactions classically defined as unidirectional may have unexpected plasticity to operate in reverse and open new avenues in biomanufacturing.

The fluorinase enzyme, the only known biocatalyst forming stable carbon-fluorine bonds, operates with extremely low efficiency-- catalyzing one reaction every 2-12 minutes. This severely limits its utility for sustainable biofluorination, and its sluggish activity remains poorly understood. We suppressed its aggregation through directed mutagenesis and elucidated the kinetic mechanism using a novel mathematical framework that fit complex kinetic and oligomerization data. This analysis revealed that >80% of enzyme molecules are inactive under standard conditions due to two dead-end pathways. A designed W50F+A279R mutant showed a 2-fold improvement in efficiency and expanded operational tolerance. Combined with mechanism-based medium optimization, the catalytic rate reached 10.1 {+/-} 3.1 min-1. Our work provides a mechanistic blueprint for fluorinase enhancement and a generalizable mathematical framework for analyzing kinetics of multimeric enzymes.

Exogenous transition metal catalysts can be combined with natural enzymes to perform designed, non-natural tandem reactions within living mammalian cells. The resulting abiotic metabolic network can process exogenous substrates to yield fluorescent or bioactive products, only in those cells that harness both catalytic systems.

Pyridoxal-5'-phosphate (PLP), the ubiquitous and ancient cofactor, plays important roles in enzymatic elimination, transamination and other reactions. The catalytic efficiency of PLP-dependent enzymes is significantly higher than that of free PLP. The recruitment of PLP from the environment by the enzymes, particularly through interactions outside the active site, is the key step determining the occurrence of PLP-mediated catalysis. However, the precise mechanism by which enzymes recruit PLP remains elusive. Methionine {gamma}-lyase (MGL), a PLP-dependent enzyme, catalyzes the degradation of L-methionine, thereby suppressing cancer cell proliferation through serum or dietary methionine depletion. Here, we report the crystal structure of yMGL, which belongs to a newly identified subgroup of cystathionine {gamma}-lyases, in complex with L-methionine and PLP. Through truncating the C-terminal domain of yMGL both in vitro and in vivo, we demonstrated that this domain, outside the canonical PLP-binding domain, is essential for the specific interaction between yMGL and PLP, as well as for efficient L-methionine catabolism. The C-terminal domain, which contains a conserved serine residue, forms a unique structural feature at the enzymes entrance that effectively traps PLP and confers PLP specificity. These findings elucidate a previously uncharacterized mechanism of PLP recruitment by MGLs and offer a structural framework to rationally design and engineer MGLs with tailored cofactor selectivity and catalytic performance. SIGNIFICANCEUnderstanding the mechanism by which enzymes recruit cofactors is essential for advancing biochemistry and developing biotechnological applications. PLP is a ubiquitous and ancient cofactor required by numerous metabolic enzymes. Despite its fundamental importance, the molecular mechanisms governing how enzymes selectively recruit and bind PLP from the environment remain poorly understood. Our structural and functional analysis of methionine {gamma}-lyases and their ancestral enzymes reveals a previously unknown PLP recruitment mechanism involving a C-terminal domain that acts as a cofactor trap outside the canonical active site. This discovery advances our understanding of enzyme-cofactor interactions and provides a rational framework for designing and engineering PLP-dependent enzymes.

Biological plastics deconstruction and upcycling have emerged as a sustainable alternative to traditional recycling technologies for plastics waste. The discovery and engineering of efficient thermostable poly(ethylene terephthalate) (PET) hydrolases has made biological PET recycling possible at scale; however, enzymes for non-PET plastics, which account for approximately 70% of all plastics produced, remain largely undiscovered. To accelerate the discovery of such enzymes, a high throughput screening (HTS) platform is needed. Here, we develop a HTS liquid-based assay to detect one of the first committed steps of polyolefin degradation, oxidation of the C-H bond to an aldehyde. We test 4-hydrazino-7-nitro-2,1,3-benxoxadiozole hydrazine (NBD-H), which reacts with generated aldehydes to form a fluorescent hydrazone, on oxidized low-density polyethylene (LDPE) films. Hydrazone generation correlated well with established carbonyl index metrics for polymer oxidation (R2 = 0.97). Moreover, we demonstrate that the probe reliably identifies LDPE-active dye decolorizing peroxidases (DyPs) that generate aldehydes on LDPE films, serving as effective screen as demonstrated by a receiver operating characteristic area under the curve of 0.95. Due to the rapid fluorescent readout and parallelization in microarray plates, this assay enables screening thousands of enzymes in 24 hours compared to time-consuming established approaches, accelerating discovery of enzymes that catalyze the first step of polyolefin biodeconstruction.

Selective radical chemistry poses fundamental challenges for modern catalysis. Non-natural photoenzymes have recently emerged as appealing systems to address these challenges by offering unmatched control over chemo-, enantio-, and substrate selectivity, yet their underlying mechanisms remain unclear. Here, we reveal the complete molecular basis of triple selectivity control in photoenzyme GkOYE-G7 through computational simulations based on multiscale multireference-quantum-mechanics/molecular-mechanics modeling and bias-exchange metadynamics. Our findings demonstrate that control emerges from reaction-level mechanisms rather than binding preferences. We discover that productive photochemistry requires a previously unkown pre-activation step involving bond elongation. Stereochemical outcomes result from reaction barrier differences, while chemoselectivity exploits early-appearing crossing points between ground and excited electronic states that commit reactions before competing pathways activate. Substrate scope follows predictable electronic-steric rules, establishing fundamental principles for engineering next-generation photoenzymes with predictable selectivity profiles.