R.B. Leveson-Gower

Many known enzymes sample huge protein conformational space, hampering structural characterization by X-ray crystallography, and preventing thus the understanding of their catalytic mechanisms. In this review, we outline that the combination of reconstructed ancestral enzymes with unconvertible substrate analogues is becoming a powerful strategy to decipher the challenging mechanisms of enzyme catalysis.

Abstract

Environmentally friendly industrial and biotech processes greatly benefit from enzyme-based technologies. Their use is often possible only when the enzyme-catalytic mechanism is thoroughly known. Thus, atomic-level knowledge of a Michaelis enzyme-substrate complex, revealing molecular details of substrate recognition and catalytic chemistry, is crucial for understanding and then rationally extending or improving enzyme-catalyzed reactions. However, many known enzymes sample huge protein conformational space, often preventing complete structural characterization by X-ray crystallography. Moreover, using a cognate substrate is problematic since its conversion into a reaction product in the presence of the enzyme will prevent the capture of the enzyme-substrate conformation in an activated state. Here, we outlined how to deal with such obstacles, focusing on the recent discovery of a Renilla-type bioluminescence reaction mechanism facilitated by a combination of engineered ancestral enzyme and the availability of a non-oxidizable luciferin analogue. The automated ancestral sequence reconstructions using FireProt^ASR provided a thermostable enzyme suited for structural studies, and a stable luciferin analogue azacoelenterazine provided a structurally cognate chemical incapable of catalyzed oxidation. We suggest that an analogous strategy can be applied to various enzymes with unknown catalytic mechanisms and poor crystallizability.

Photocaged glutamic acid analogues are genetically encoded into proteins for the first time, opening up new opportunities for directly and effectively generating photoactivatable proteins.

Abstract

Genetically replacing an essential residue with the corresponding photocaged analogues via genetic code expansion (GCE) constitutes a useful and unique strategy to directly and effectively generate photoactivatable proteins. However, the application of this strategy is severely hampered by the limited number of encoded photocaged proteinogenic amino acids. Herein, we report the genetic incorporation of photocaged glutamic acid analogues in E. coli and mammalian cells and demonstrate their use in constructing photoactivatable variants of various fluorescent proteins and SpyCatcher. We believe genetically encoded photocaged Glu would significantly promote the design and application of photoactivatable proteins in many areas.

Unspecific peroxygenases (UPOs) have recently gained attraction as versatile oxyfunctionalization catalysts. One shortcoming, however, is their susceptibility towards the co-substrate hydrogen peroxide. As a solution, the concept of light-dependent UPO biocatalysis with genetically encoded flavin-containing photosensitizer proteins for in situ hydrogen peroxide production is introduced.

Abstract

Fungal unspecific peroxygenases (UPOs) have gained substantial attention for their versatile oxyfunctionalization chemistry paired with impressive catalytic capabilities. A major drawback, however, remains their sensitivity towards their co-substrate hydrogen peroxide, necessitating the use of smart in situ hydrogen peroxide generation methods to enable efficient catalysis setups. Herein, we introduce flavin-containing protein photosensitizers as a new general tool for light-controlled in situ hydrogen peroxide production. By genetically fusing flavin binding fluorescent proteins and UPOs, we have created two virtually self-sufficient photo-enzymes (PhotUPO). Subsequent testing of a versatile substrate panel with the two divergent PhotUPOs revealed two stereoselective conversions. The catalytic performance of the fusion protein was optimized through enzyme and substrate loading variation, enabling up to 24300 turnover numbers (TONs) for the sulfoxidation of methyl phenyl sulfide. The PhotUPO concept was upscaled to a 100 mg substrate preparative scale, enabling the extraction of enantiomerically pure alcohol products.

Non-canonical amino acids (ncAAs) are prized building blocks in the synthesis of natural products, designer peptides and drug molecules. Despite their general utility, the complex structure of these molecules still presents an enormous challenge for chemical synthesis. Here, we develop a one-pot chemoenzymatic approach for the construction of azacyclic ncAAs with multiple substitutions and various ring sizes. A promiscuous transaminase was identified to convert a wide range of diketoacids to the corresponding α-amino acids. A spontaneous cyclic imine formation was followed by a stereocontrolled chemical reduction to generate the corresponding products in one-pot with high stereoselectivity. More than twenty azacyclic ncAAs were successfully prepared with this approach. This work demonstrates the value of developing hybrid biocatalytic-chemocatalytic approaches to privileged small molecule motifs.

Nature, Published online: 16 August 2023; doi:10.1038/d41586-023-02585-7

Efforts to replicate the material have pieced together the puzzle of why it displayed superconducting-like behaviours.

Science, Volume 381, Issue 6659, Page 754-760, August 2023.

Nature Chemistry, Published online: 10 August 2023; doi:10.1038/s41557-023-01287-x

The design and improvement of enzymes based on physical principles remain challenging. Now, the vibrational Stark effect has been used to demonstrate how an electrostatic model can unify the catalytic effects of distinct chemical forces in a quantitative manner and guide the design of enzyme variants that outperform their natural counterpart.

Nature, Published online: 08 August 2023; doi:10.1038/s41586-023-06507-5

Bonding wood with uncondensed lignins as adhesives

Nature, Published online: 02 August 2023; doi:10.1038/d41586-023-02457-0

Massive vertebrae and other fossilized remains found in Peru point to an Eocene-epoch beast of colossal proportions.

Proceedings of the National Academy of Sciences, Volume 120, Issue 31, August 2023.

Nature Reviews Chemistry, Published online: 31 July 2023; doi:10.1038/s41570-023-00523-9

To combat worsening environmental crises, chemistry needs a redesign. We see the need for a triple focus on efficiency, safety and circularity as a prerequisite for chemistry to serve sustainability and ensure that essential chemical products and processes are waste-free, functional and safe for both humans and the environment.

Nature Catalysis, Published online: 31 July 2023; doi:10.1038/s41929-023-00994-5

The generation of nitrogen-centred radicals and their subsequent reaction with control of stereoselectivity is a difficult task in synthetic chemistry. Now, the photoenzymatic production of nitrogen-centred radicals and their use in challenging enantioselective intermolecular radical hydroaminations is reported.

Isonitrileproline (Inp) is introduced as the first isonitrile-containing amino acid for solid-phase peptide synthesis. The conformation directing properties of the isonitrile group and its effect on the collagen triple helix were elucidated. The value of Inp for peptide derivatization was showcased by a chemoselective ligation with a functional chlorooxime.

Abstract

Functional groups that allow for chemoselective and bioorthogonal derivatization are valuable tools for the labelling of peptides and proteins. The isonitrile is such a group but synthetic methods for its incorporation into peptides by solid-phase peptide synthesis are not known. Here, we introduce (4S)- and (4R)-isonitrileproline (Inp) as building blocks for solid-phase peptide synthesis. Conformational studies of (4S)- and (4R)-Inp and thermal stability analysis of Inp-containing collagen triple helices revealed that the isonitrile group exerts a stereoelectronic gauche effect. We showcase the value of Inp for bioorthogonal labelling by derivatization of Inp-containing collagen model peptides (CMPs). Dual labelling with a pair of bioorthogonal reactions of a CMP containing Inp and azidoproline residues further highlights the versatility of the new isonitrile-containing amino acids.

Science, Volume 381, Issue 6656, Page 444-451, July 2023.

Bioelectrocatalytic synthesis represents an exciting merger of selective biocatalysis and green electrochemical methods for the synthesis of value-added chemicals. In this review we introduce the key concepts and vital applications of both enzymatic and microbial electrosynthetic systems.

Abstract

Bioelectrocatalytic synthesis is the conversion of electrical energy into value-added products using biocatalysts. These methods merge the specificity and selectivity of biocatalysis and energy-related electrocatalysis to address challenges in the sustainable synthesis of pharmaceuticals, commodity chemicals, fuels, feedstocks and fertilizers. However, the specialized experimental setups and domain knowledge for bioelectrocatalysis pose a significant barrier to adoption. This review introduces key concepts of bioelectrosynthetic systems. We provide a tutorial on the methods of biocatalyst utilization, the setup of bioelectrosynthetic cells, and the analytical methods for assessing bioelectrocatalysts. Key applications of bioelectrosynthesis in ammonia production and small-molecule synthesis are outlined for both enzymatic and microbial systems. This review serves as a necessary introduction and resource for the non-specialist interested in bioelectrosynthetic research.

Introducing new abiotic chemistry into living cells is a grand challenge in the field of chemical biotechnology. Here, we show that phosphate and/or biogenic amines mediate the non-enzymatic α-methylenation of butyraldehyde under biocompatible reaction conditions and can be interfaced with butyraldehyde biosynthesis from D-glucose in the bacterium Escherichia coli.

Abstract

Small molecule organocatalysts are abundant in all living organisms. However, their use as organocatalysts in cells has been underexplored. Herein, we report that organocatalytic aldol chemistry can be interfaced with living Escherichia coli to enable the α-methylenation of cellular aldehydes using biogenic amines such as L-Pro or phosphate. The biocompatible reaction is mild and can be interfaced with butyraldehyde generated from D-glucose via engineered metabolism to enable the production of 2-methylenebutanal (2-MB) and 2-methylbutanal (2-MBA) by anaerobic fermentation, and 2-methylbutanol (2-MBO) by whole-cell catalysis. Overall, this study demonstrates the combination of non-enzymatic organocatalytic and metabolic reactions in vivo for the sustainable synthesis of valuable non-natural chemicals that cannot be accessed using enzymatic chemistry alone.