Biocatalysis@TUDelft

A halohydrin dehalogenase from Novosphingobium resinovorum (HHDHnsr) is characterized with high α-regioselectivity in azide-mediated epoxide ring-opening reactions. Phylogenetic analysis classifies HHDHnsr as a G-type enzyme, designated as HheG4. By combining HheG4 with β-regioselective HHDHs, a parallel kinetic resolution strategy is proposed for the synthesis of enantiomerically enriched β-azidoalcohols from racemic epoxides.

Halohydrin dehalogenases are highly valuable biocatalysts for chiral compound synthesis due to their diverse catalytic capabilities and inherent stereoselectivity. Historically, research on halohydrin dehalogenases is confined to a limited number of enzymes. However, with the advent of advanced gene mining methods, a growing number of halohydrin dehalogenases have been discovered and characterized, revealing a diverse array of new structures and catalytic functions. While halohydrin dehalogenases predominantly exhibit β-regioselectivity in the ring-opening of styrene oxides, a few α-regioselective halohydrin dehalogenases have also been identified. In this study, a new α-regioselective halohydrin dehalogenase, HHDHnsr, is purified and analyzed, and its application in the azide-mediated α-regioselective azidolysis of styrene oxide is explored. The findings highlight the potential of HHDHnsr as α-regioselective biocatalyst for the asymmetric synthesis of β-azidoalcohols, offering valuable insights for the future identification of α-regioselective HHDHs in enzyme mining.

Recombinant protein expression is central to biotechnology’s application. However, not all proteins can be expressed in all organisms, and, given the vast experimental space, it can be challenging to identify the conditions that will yield successful protein expression. The field lacks a predictive model of soluble protein expression that could replace laborious experimental trial and error. Here, we discuss the state of the field and identify the lack of large, high-fidelity datasets as the primary bottleneck to progress. We outline a proposed path toward an extensible experimental platform for collecting soluble overexpression data across organisms. We suggest that the resulting data should be used to train predictive models of protein expression toward answering the question: can protein expression be solved?

Recent developments in DNA synthesis and sequencing have allowed the construction of comprehensive gene variant libraries and their functional analysis. Achieving high-replication and thorough mutation characterization remains technically and financially challenging for long genes. Here, we developed an efficient, affordable and scalable library construction approach that relies on low-cost DNA synthesis and standard cloning technologies, which will increase accessibility to systematic mutational studies and help advance the field of protein science.

Multiheme cytochromes c are versatile enzymes which contribute to key steps in diverse biogeochemical cycles of elements and are also key players in microbial electrochemical technologies. We previously showed that these enzymes evolve by grafting and pruning of cytochrome c modules. Here, we extended our analysis and found that grafting can also involve the incorporation of domains of other protein families and of small peptide sequences that affect the heme coordination environment. We show that the addition of hemes to multiheme cytochromes c occurs exclusively by integrating heme-binding peptides and that gain of hemes is equally probable along the protein sequence. By contrast, heme loss occurs by the loss of heme-binding peptides and the accumulation of point mutations. Notably, heme-binding motif loss is disproportionately more prevalent at the position nearer to the N-terminus. This observation contrasts with the general trend observed in proteins, which are usually more conserved at the N-terminus, and likely reflects the way in which multiheme cytochromes c are assembled. Overall, this work completes the picture of how multiheme cytochromes c evolved to achieve the diversity of structures and functions found in nature, and sets them apart from other proteins with respect to the drivers for their evolution. This has contributed to the diversity of roles that they play in various biogeochemical cycles and has implications for engineering artificial variants to enhance biotechnological applications.

[NiFe] hydrogenases are a family of enzymes that can be used to produce biofuel, thus making them important for industrial applications. In this work, we utilized unbiased molecular dynamics (UMD) simulations to capture binding and unbinding events of the substrate, H2, to and from the [NiFe] hydrogenases from two different organisms. We obtained multiple (un)binding events and reproduced experimental association rate constants. We observed symmetry between the binding and unbinding pathways used by H2 to access or leave the catalytic site. Moreover, we found that the main bottleneck for ligand binding, the distance between residues V74 and L122, can shift between two states with different bottleneck widths, a feature which can be exploited to modulate the access of small molecules to the catalytic site. The pathway probabilities presented here can be used to benchmark enhanced sampling methods which investigate protein-ligand binding.

Photobiocatalysis enables remarkable synthetic transformations by combining the exquisite stereoselectivity of enzymes with the mild generation of high-energy intermediates by photocatalysis, but practical applications remain limited due to enzyme photodamage. The deracemization of secondary alcohols is a key model reaction for photobiocatalytic protocols due to the importance of the enantioenriched products. However, current strategies rely on the temporal separation of catalytic cycles to circumvent incompatibilities, precluding photobiocatalytic transformations that require the in situ generation of reactive intermediates. We report a single-step cyclic deracemization protocol by combining a water-soluble photocatalyst (sodium anthraquinone-2-sulfonate) with a promiscuous alcohol dehydrogenase (Geotrichum candidum acetophenone reductase) encapsulated in lyophilized microbial whole-cells. Insights into enzyme selectivity and system dynamics from molecular docking and kinetic modeling guided the optimization of the multi-component system. Our approach represents a modular and generalizable strategy for developing photobiocatalytic cascades operating under mutually compatible conditions, wherein spatial separation mitigates photodamage and enables simultaneous dual catalytic turnover.

Visible light photocatalysis that exploits the reactivity of molecules at their excited state has induced a paradigm shift in organic synthesis by enabling unique chemical transformations, but controlling their enantioselectivity has proven difficult. A promising strategy involves linking a synthetic transition metal photocatalyst within the chiral architecture of a biomolecule to create a highly selective artificial photoenzyme. However, such a biohybrid system that combines the merits of biocatalysis and metallo-photocatalysis to promote abiological reactions fueled by visible light with high enantioselectivity is still unknown. Here, we report on an artificial metallo-photoDNAzyme resulting from covalently anchoring a blue light absorbing iridium-based photocatalyst within a double-stranded DNA helix that exhibits efficient triplet-triplet energy transfer and high levels of enantioselectivity in [2+2] intramolecular cycloadditions.

Methanogenic archaea contribute 1-2 Gt of methane annually, impacting both global carbon cycling and climate. Central to their energy metabolism is a membrane-bound, sodium-translocating methyltransferase complex: the N-tetrahydromethanopterin:CoM-S-methyltransferase (Mtr complex), which catalyzes the methyl transfer between two methanogen specific cofactors. This exergonic methyl transfer step is coupled with a vectorial sodium ion transport from the cytoplasm to the cell exterior and is the only energy conserving step in hydrogenotrophic methanogenesis. Here, we present a 2.1 [A] single-particle cryo-EM structure of the full Mtr complex from Methanosarcina mazei. Our structural model encompasses the entire complex, reveals the arrangement of archaeal phospholipids, the architecture of the sodium ion binding site, and the structure and interactions of all catalytic subunits. Most strikingly, we discover and characterize MtrI, a previously unannotated small open reading frame (small ORF), encoded protein (<100 aa) conserved across the order of Methanosarcinales. MtrI binds to the cytoplasmic domain of MtrA in response to oxygen exposure, suggesting a role in oxygen stress response and protection. By binding on top of the sodium channel and anchoring to the cobamide cofactor in MtrAs cytoplasmic domain, MtrI might prevent sodium leakage and inhibit MtrA-CoM turnover. These findings offer new insights into methanogen energy conservation and uncover a potential adaptive response to oxygen exposure, expanding our understanding of methanogen survival strategies under oxidative stress.

Fortification of FeS clusters in Ni─Fe CO dehydrogenase (Carboxydothermus hydrogenoformans) reshapes this anaerobic enzyme into an air-viable variant through multilayered O₂ tunnel sealing. The engineered enzyme retains structural integrity and catalytic efficiency in ambient air, offering a practical solution for industrial CO/CO₂ bioconversion processes without the need for strict anaerobic conditions.

Abstract

The inherent O₂ sensitivity of Ni─Fe carbon monoxide dehydrogenases (CODHs), crucial for rapid CO to CO₂ interconversion, presents substantial challenges for industrial application. Transforming CO/CO₂, a prevalent anthropogenic air pollutant, into valuable carbon chemicals either directly or through intermediate steps via biocatalytic methods offers a promising pathway to achieve net-zero emissions across industries and the environment. However, completely eliminating oxygen from industrial biotransformations, especially under ambient conditions, is exceedingly onerous. Here, we engineered variants of the CODH2 from Carboxydothermus hydrogenoformans (ChCODH2) with dual blocking at both the O₂ entrance and near the active site, effectively sealing the tunnel against atmospheric O₂ levels (20%). The O₂-tunnel engineered A559W/V610H variant demonstrated a marked improvement in air stability, with a half-life of 24.6 h compared to the wild type's 2.4 h. Crystallographic snapshots of this air-viable variant after 24 h of exposure revealed the robust integrity of the fortified FeS and NiFeS clusters. Additionally, electro-enzymatic reactions corroborated its CO/CO₂ conversion capability even in ubiquitous air. These findings, which address the O₂ sensitivity of anaerobic enzymes caused by O₂-induced metal cluster collapse, enhance their potential for biological CO/CO₂ transformations in O₂-rich environments, thereby broadening their industrial viability and applicability.

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

Despite significant progress in protein design, enzyme catalysis remains a major challenge. Here, we present AI.zymes, a computational platform for evolutionary enzyme design. Through iterative rounds of design and screening, AI.zymes integrates cutting-edge bioengineering tools (ProteinMPNN, Rosetta, ESMFold, FieldTools, etc.) into a modular pipeline. AI.zymes was experimentally benchmarked by enhancing the catalytic activity of a model enzyme 7.7-fold.

Abstract

The ability to create new-to-nature enzymes would substantially advance bioengineering, medicine, and the chemical industry. Despite recent breakthroughs in protein design and structure prediction, designing novel biocatalysts remains challenging. Here, we present AI.zymes, a modular platform integrating cutting-edge protein engineering algorithms within an evolutionary framework (https://github.com/bunzela/AIzymes). By combining bioengineering tools such as Rosetta, ESMFold, ProteinMPNN, and FieldTools in iterative rounds of design and selection, AI.zymes can optimize a broad range of catalytically relevant properties. In addition to enhancing transition state affinity and protein stability, AI.zymes can also improve properties that are not targeted by the employed design algorithms. For instance, AI.zymes can enhance electrostatic catalysis by iteratively selecting variants with stronger catalytic electric fields. Benchmarking AI.zymes on the promiscuous Kemp eliminase activity of ketosteroid isomerase led to a 7.7-fold activity increase after experimentally testing just 7 variants. Due to its modularity, AI.zymes can readily incorporate emerging design algorithms, paving the way for a unifying framework for enzyme design.

Nature, Published online: 02 June 2025; doi:10.1038/d41586-025-01674-z

Most proteins are left-handed, but scientists have found an ancient molecule that works in both mirror-image forms.