Biocatalysis@TUDelft

A unique chemoenzymatic synthesis route toward (S)-baclofen is presented, which highlights a hydroformylation and biocatalysis step toward the synthesis of such a challenging β-chiral amino acid. The shown methodology exhibits high conversions and enantiomeric excess and is transferable to other β-chiral amines and amino acids.

This study explores the chemoenzymatic synthesis of (S)-baclofen, which involves a sequential combination of transition metal catalysis and biocatalysis. The synthesis approach starts from a readily accessible cinnamic acid ester that is converted using a rhodium-based hydroformylation catalyst toward the corresponding chiral aldehyde. This compound is subsequently converted via a transaminase-catalyzed reaction system that yields the desired β-chiral amino acid ester and the final free β-chiral amino acid ( S )-baclofen after a simple hydrolysis reaction. This synthesis concept does provide high atom efficiency and does not require an additional chiral resolution step of a racemic product.

The protein LmrR is a versatile scaffold to design artificial metalloenzymes. Here, we show that the M8D/A92E (DE) mutations that result in a much more efficient catalyst, destabilize the protein dimerization interface and alter the conformation landscape when binding metal cofactor and substrates are shown. These results highlight the intricate relations between protein, metal cofactor, and substrates in defining catalytic efficiency.

Artificial metalloenzymes (ArM) hold great potential for the sustainable catalysis of complex new-to-nature reactions. To efficiently improve the catalytic efficacy of ArMs, a rational approach is desirable, requiring detailed molecular insight into their conformational landscape. Lactococcal multidrug resistance regulator (LmrR) is a multipurpose ArM scaffold protein that, when bound to the Cu(II)-phenanthroline cofactor, catalyzes the Friedel–Crafts alkylation (FCA) of indoles. Previously, the M8D and A92E mutations are found to increase the efficiency of this reaction, but a molecular explanation has been lacking. The impact of these two activating mutations on the conformational landscape of LmrR in its apo, cofactor- and substrate-bound states is determined. The mutations cause a marked destabilization of the dimerization interface, resulting in a more open central hydrophobic cavity and a dynamic equilibrium between dimer and monomer LmrR is found. While mutant and wild-type have similar pocket conformation in the cofactor-bound state, the mutant shows a distinct interaction with the substrate. Our results suggest that increased retention of the catalytic cofactor and widened plasticity improve the activity of the mutant. Ultimately, these results help elucidating the intricate relationships between conformational dynamics of the protein scaffold, cofactor, and substrates that define catalytic activity.

Biocatalysis has proven to be a powerful and efficient method for synthesizing oxindoles, leveraging enzymes such as metalloenzymes, halohydrin dehalogenases, and oxygenases, among others. Additionally, the strategic integration of enzymatic reactions with chemical techniques, including photo-enzyme and electro-enzyme catalysis, creates new opportunities to enhance the biocatalytic synthesis of oxindoles.

Abstract

Oxindoles have demonstrated significant applications in medicinal chemistry and synthetic organic chemistry, making the development of efficient synthetic strategies for their preparation highly important. In addition to traditional chemical synthesis methods, biocatalysis has emerged as a powerful and effective approach for the synthesis of oxindoles. Biocatalysis offers unique advantages, such as mild reaction conditions, high selectivity, and environmental compatibility. In this review, we concentrate on biocatalytic methods for oxindole synthesis, encompassing both the formation of the oxindole core and the synthesis of its derivatives. It aims to serve as an essential reference for researchers in synthetic chemistry, medicinal chemistry, and biocatalysis, encouraging further innovation in this significant area of study.

Applied and Environmental Microbiology, Volume 91, Issue 7, July 2025.

Nature, Published online: 18 June 2025; doi:10.1038/d41586-025-01897-0

Computer approach creates synthetic enzymes 100 times more efficient than those designed by AI.

Nature, Published online: 18 June 2025; doi:10.1038/s41586-025-09136-2

We present a computational approach to the design of high-efficiency enzymes with catalytic parameters comparable to natural enzymes, enabling programming of stable, high-efficiency, new-to-nature Kemp elimination enzymes through minimal experimental effort.

Nature Chemistry, Published online: 16 June 2025; doi:10.1038/s41557-025-01852-6

Target-oriented syntheses of natural products usually require the design of individualized routes that are tailor-made for the specific targets. Now, a strategy that runs counter to this conventional wisdom has been achieved. Exploiting a biocatalytically installed alcohol, several abiotic skeletal rearrangements have been designed to prepare three structurally disparate terpenoid natural products.

Cyanide is widely used in industries due to its strong affinity for metals, a property that also underlies its potent toxicity. Industries therefore must reduce cyanide concentration in wastewater final disposal. Physical, chemical, and biological methods have been developed for this purpose; however, knowledge about the structure of enzymes involved in cyanide degradation remains limited. Structural characterization of these proteins could facilitate the development of more efficient enzymes with enhanced bioremediation potential. Here, we present the single-particle cryo-electron microscopy structures of a cyanide dihydratase from Bacillus safensis and a cyanide hydratase from Gloeocercospora sorghi at 2.2 [A] and 2.0 [A] resolution, respectively. We provide a comprehensive description and comparative analysis of these structures alongside all previously experimentally determined nitrilase structures. Importantly, our full-length structures reveal new structural features in the C-terminal as well as specific intermolecular interactions between protomer interfaces and within the helix lumen. Finally, our findings offer insights into the possible reaction mechanisms of these two enzymes.

Nucleoside monophosphates (NMPs) are the first nucleotides required for the conversion of nucleosides into nucleoside triphosphates. Biocatalysis can offer an environmentally sustainable route to NMPs, but process development is a key consideration towards ensuring full efficiency. Herein we demonstrate a continuous biocatalytic platform using a promiscuous nucleoside kinase, DmDNK, to enable scalable production of a panel of non-natural NMPs in flow. The system takes advantage of enzyme immobilization to enable a dual-enzyme cascade for in situ ATP recycling, improved enzymatic productivity versus batch and accessing multi-milligram scale production of several important NMPs; isolated yields exceed 70% for 2′-deoxy-2′-fluorouridine, cytarabine and gemcitabine NMPs, with space time yields all over 22.5 g L-1 h-1. This platform provides a basis for the expansion of biocatalytic NMP production towards a sustainable delivery of these essential building blocks, including those underpinning to therapeutically relevant nucleoside triphosphates.

Traditional biocatalytic cascades typically require discrete enzymes for each synthetic step. Here, we report unprecedented trifunctional imine reductases (IRED) that conduct three sequential transformations—alkene reduction, intramolecular reductive amination, and imine reduction—all within a single catalytic cycle. This elegant single-enzyme catalytic system directly transforms linear substrates into enantiomerically pure 2-aryl pyrrolidines via a concerted cascade without intermediate isolation. Combining density functional theory (DFT) calculations and mechanistic studies, we elucidate how the IRED achieves step-selective catalysis. Our findings establish a proof-of-concept for simplifying complex biocascades using multifunctional enzymes, offering a powerful strategy to streamline synthetic pathways.

Enzyme specificity defines function: aminophenol oxidase uniquely converts aminophenols into nitrosophenols, whereas tyrosinase transforms them into quinone imines. This distinction is governed by a key amino acid near the copper(A) active site. In their Research Article (e202501560), Annette Rompel et al. demonstrated that a single amino acid exchange—swapping asparagine in SgGriF with isoleucine in SzTYR—can effectively invert the enzymes’ functions.

Peptidomelanin is a fungal biopolymer secreted during the germination of Aspergillus niger melanoliber spores. Peptidomelanin is composed of an insoluble L-DOPA-derived core polymer surrounded and solubilized by short, heterogenous peptide chains. In this work, we report the identification and biochemical characterization of BroSCO (gene ID: ABHI18_005222), a broad spectrum copper oxidase responsible for synthesizing peptidomelanin. Our model of BroSCO possesses a type 3 di-copper centre housed in a large, solvent accessible catalytic center that can accommodate substrates possessing a range of sizes. BroSCO oxidizes thiol groups from cysteine and cysteinylated peptides, allowing them to copolymerize with L-DOPA, forming peptidomelanin in the latter case. Our identification of BroSCO as the enzyme responsible for synthesizing peptidomelanin lead to the characterization of a previously unknown biochemical pathway, expanding our understanding of fungal biochemistry and melanogenesis.

Phosphates are essential for all forms of life, playing key roles in DNA/RNA, signaling, energy storage and transfer, and biosynthetic processes. We conducted a quantitative analysis of nucleoside triphosphate (NTP) processing enzymes across all enzymatic reactions, revealing their dominance in phosphate reactivity with ATP as the most prevalent substrate. Two main reaction types occur predominantly: cleavage resulting in (i) pyrophosphate or (ii) phosphate release/transfer. The large majority of NTP processing enzymes require divalent Mg2+ ions in a mechanistically analogous manner. Despite their importance, the precise coordination of metal ions in the catalytically competent active site structure in many NTP processing enzymes remains elusive. We hypothesized that efficient phosphate processing requires specific metal ion coordination, facilitating the catalytic reaction. By examining a vast dataset of crystallographic structures, we identified a universal "Mg-pinch" motif, confirming our structural hypothesis for almost all NTP processing enzymes. We postulated that the Mg2+ ion should coordinate both phosphates that are involved in the cleaved P-O bond. We present a comprehensive analysis of NTP processing superfamilies across all species, determining distinct enzyme active site structures. We highlight exceptional cases and propose challenging superfamilies that lack sufficient structural data to determine precise active site coordination. Our quantum mechanical calculations based on DFT-based QM/MM with full electrostatic embedding revealed the essential electrostatic polarization effects of the Mg²⁺ ion as key determinants of their role in the enzyme catalysis. The Mg-pinch motif provides a mechanistic framework for understanding the catalytic role of metal ions in NTP processing. Our findings offer insights into enzyme evolution, provide a basis for rational enzyme engineering, and could inform the development of novel therapeutics targeting NTP processing enzymes.

Science, Volume 388, Issue 6752, June 2025.