Search

A generative artificial intelligence-powered method enables de novo design of highly active enzymes based on information about the geometry of residues in the active site, without requiring protein backbone or sequence information.

A haem–carbenoid has been proposed to be involved in abiological enzymatic reactions. Now, Hilvert and co-workers provide crystallographic evidence for a haem–carbenoid intermediate as the reactive species in an olefin cyclopropanation reaction catalysed by an artificial metalloenzyme.

Formate produced by synthetic methylotrophic E. coli can lead to carbon loss and negatively impact bioproduction efficiency. Here, the authors report the production of formate as a widespread property of NAD-dependent methanol dehydrogenases and identify Mdhs without this overoxidation activity.

Nonribosomal peptide synthetases (NRPSs) produce vital natural products but have proven recalcitrant to biosynthetic engineering. Now, a combination of yeast surface display and fluorescence-activated cell sorting (FACS) has been used to reprogram an L-Phe-incorporating module for β-Phe. The resulting module is highly selective and functions efficiently in NRPS pathways.

Protein cages are used for encapsulation of macromolecules such as proteins and nucleic acids. Here, the authors report a strategy to encapsulate poorly soluble small molecules within porous protein cages through capsid-templated micelle formation and show that the resulting lipoprotein-like cages enhance the cellular delivery of these molecules.

Ready access to sugars in which the various hydroxyl groups are differentially protected will be of benefit in the production of vaccines, antibiotics and drugs. Here, a chemoenzymatic method that provides a direct route to such protected sugars is described.

Self-assembling proteins that form capsid-like structures act as molecular containers for diverse cargoes. Here, the authors solve the cryo-EM structures of lumazine synthase shells, and show that supercharged mutants form expanded assemblies, indicating that electrostatics can be exploited to engineer cage architecture.

A stimulus-responsive approach for recapitulating nonviral nucleocapsid assembly on demand under controlled conditions provides a robust platform for applications in synthetic biology and mRNA nanomedicine.

An artificial aldolase has been optimized using an ultrahigh-throughput microfluidic screening assay. The evolved enzyme exhibits excellent stereoselectivity and broad substrate scope. Structural studies suggest that a Lys-Tyr-Asn-Tyr catalytic tetrad, which emerged during directed evolution, is responsible for the >10⁹ rate enhancement achieved by this catalyst.

Nonribosomal peptide synthetases produce valuable natural products but are challenging to engineer. Yeast surface display and fluorescence-activated cell sorting have now been combined to reprogram a condensation domain to recognize a noncanonical lipid substrate. This methodology may facilitate molecular tailoring of many biosynthetic assembly lines.

A de novo designed zinc-binding protein has been converted into a highly active, stereoselective catalyst for a hetero-Diels–Alder reaction. Design and directed evolution were used to effectively harness Lewis acid catalysis and create an enzyme more proficient than other reported Diels–Alderases.

By enriching productive mutational paths, a Kemp eliminase that speeds up proton abstraction >10⁸-fold was developed in only five evolution rounds. Recombining it with a variant differing by 29 substitutions revealed the underlying fitness landscape.

Directed evolution typically requires extensive screening. This work presents an ultrahigh-throughput microfluidic assay, based on a coupled reaction and fluorescence-activated droplet sorting, enabling a 960-fold activity improvement of an amine oxidase for a non-natural substrate in a single round.

A previously designed enzyme used a reactive lysine to initiate cleavage of a carbon-carbon bond. Directed evolution of this construct now shows a drastic reorganization of the active site to use an alternative catalytic lysine and suggests considerations for future design efforts.

Cyanophycin synthetase CphA1 polymerizes Asp and Arg into the nitrogen reserve polymer cyanophycin using two active sites. Sharon et al. show CphA1 has a cryptic 3rd active site that cleaves cyanophycin into primers for self-sufficient biosynthesis.

Structures of three cyanophycin synthetases reveal how the constituent glutathione synthetase and muramyl ligase-like domains cooperate to make cyanophycin, a poly-aspartate chain with arginine residues attached to the sidechains by isopeptide bonds.

Established bacterial glycoengineering platforms limit access to protein and glycan substrates. Here the authors design a cytoplasmic protein glycosylation system, Glycoli, to generate a variety of multivalent glycostructures.

Computationally designed enzymes can be substantially improved by directed evolution. Now, it has been shown that evolution can introduce a dynamic network that selectively tightens the transition-state ensemble, giving rise to a negative activation heat capacity. Targeting such transition state conformational dynamics may expedite de novo enzyme creation.

Recent progress in computational enzyme design, active site engineering and directed evolution are reviewed, highlighting methodological innovations needed to deliver improved designer biocatalysts.

A computationally designed enzyme that was evolved to accelerate a chemical reaction 6 × 10⁸-fold approaches the exceptional efficiency of highly optimized natural enzymes and provides valuable lessons for the creation of more sophisticated artificial catalysts.

Technologies are now in place to obtain large amounts of data for systems biology approaches. What are the most suitable technologies for fast, accurate and high-throughput data collection? And following data collection, how should these data be analysed and validated?