Computational design of highly active de novo enzymes

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 3, 2024

Custom designed enzymes can further enhance the use of biocatalysts in industrial biotransformations, thereby helping to tackle biotechnological challenges 21st century. We present rotamer inverted fragment finder - diffusion (Riff-Diff) a hybrid machine learning and atomistic modeling strategy for scaffolding catalytic arrays de novo protein backbones with custom substrate pockets. used Riff-Diff scaffold tetrad capable efficiently catalyzing retro-aldol reaction. Functional designs exhibit high fold diversity, pockets similar natural enzymes. Some thus generated show activities rivaling those optimized by in-vitro evolution. The design can, principle, be applied any catalytically competent amino acid constellation. These findings are paving way address factors practical applicability catalysts processes shed light on fundamental principles enzyme catalysis.

Language: Английский

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On synergy between ultrahigh throughput screening and machine learning in biocatalyst engineering

Faraday Discussions, Journal Year: 2024, Volume and Issue: 252, P. 89 - 114

Published: Jan. 1, 2024

Protein design and directed evolution have separately contributed enormously to protein engineering. Without being mutually exclusive, the former relies on computation from first principles, while latter is a combinatorial approach based chance. Advances in ultrahigh throughput (uHT) screening, next generation sequencing machine learning may create alternative routes engineered proteins, where functional information linked specific sequences interpreted extrapolated

Language: Английский

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Computational protein design

Nature Reviews Methods Primers, Journal Year: 2025, Volume and Issue: 5(1)

Published: Feb. 27, 2025

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Emergence of binding and catalysis from a designed generalist binding protein

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 31, 2025

Abstract The evolution of proteins that bind to small molecules and catalyze chemical transformations played a central role in the emergence life. While natural have finely tuned affinity for their primary ligands, they also often weak affinities other molecules. These interactions serve as starting points new specificities functions. Inspired by this concept, we determined ability simple de novo protein set diverse (< 300 Da) crystallographic fragment screening. We then used information design one variant binds fluorogenic molecule another acts highly efficient Kemp eliminase enzyme. Collectively, our work illuminates how novel functions can emerge from existing proteins.

Language: Английский

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Droplet microfluidic screening to engineer angiotensin-converting enzyme 2 (ACE2) catalytic activity

Journal of Biological Engineering, Journal Year: 2025, Volume and Issue: 19(1)

Published: Feb. 3, 2025

Angiotensin-Converting Enzyme 2 (ACE2) is a crucial peptidase in human peptide hormone signaling, catalyzing the conversion of Angiotensin-II to Angiotensin-(1–7), which activates Mas receptor and elicits vasodilation, increased blood flow, reduced inflammation, decreased pathological tissue remodeling. This study leverages protein engineering enhance ACE2's therapeutic potential for treating conditions such as respiratory viral infections, acute distress syndrome, diabetes. Surrogate substrates used traditional high-throughput screening methods peptidases often fail accurately mimic native substrates, leading less effective enzyme variants. Here, we developed an ultra-high-throughput droplet microfluidic platform screen on substrates. Our assay detects substrate cleavage via free amino acid release, providing precise measurement biologically relevant activity. Using this new platform, screened large library ACE2 variants, identifying position 187 hotspot enhancing Further focused revealed K187T variant, exhibited fourfold increase catalytic efficiency (kcat/KM) over wild-type ACE2. work demonstrates microfluidics engineering, offering robust accessible method optimize properties clinical applications.

Language: Английский

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From specialization to broad adoption: Key trends in droplet microfluidic innovations enhancing accessibility to non-experts

Biomicrofluidics, Journal Year: 2025, Volume and Issue: 19(2)

Published: March 1, 2025

Droplet microfluidics has emerged as a versatile and powerful tool for various analytical applications, including single-cell studies, synthetic biology, directed evolution, diagnostics. Initially, access to droplet was predominantly limited specialized technology labs. However, the landscape is shifting with increasing availability of commercialized manipulation technologies, thereby expanding its use non-specialized Although these commercial solutions offer robust platforms, their adaptability often constrained compared in-house developed devices. Consequently, both within industry academia, significant efforts are being made further enhance robustness automation droplet-based not only facilitate transfer non-expert laboratories but also reduce experimental failures. This Perspective article provides an overview recent advancements aimed at accessibility systems enabling complex manipulations. The discussion encompasses diverse aspects such generation, reagent addition, splitting, washing, incubation, sorting, dispensing. Moreover, alternative techniques like double emulsions hydrogel capsules, minimizing or eliminating need microfluidic operations by end user, explored. These developments foreseen integration intricate manipulations users in workflows, fostering broader faster adoption across scientific domains.

Language: Английский

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Fluorogenic, Subsingle-Turnover Monitoring of Enzymatic Reactions Involving NAD(P)H Provides a Generalized Platform for Directed Ultrahigh-Throughput Evolution of Biocatalysts in Microdroplets

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: March 24, 2025

Enzyme engineering and discovery are crucial for a sustainable future bioeconomy. Harvesting new biocatalysts from large libraries through directed evolution or functional metagenomics requires accessible, rapid assays. Ultrahigh-throughput screening formats often require optical readouts, leading to the use of model substrates that may misreport target activity necessitate bespoke synthesis. This is particular challenge when glycosyl hydrolases, which leverage molecular recognition beyond glycosidic bond, so complex chemical synthesis would have be deployed build fluoro- chromogenic substrate. In contrast, coupled assays represent modular "plug-and-play" system: any enzyme–substrate pairing can investigated, provided reaction produce common intermediate links catalytic detection cascade readout. Here, we establish producing fluorescent readout in response NAD(P)H via glutathione reductase subsequent thiol-mediated uncaging reaction, with low nanomolar limit plates. Further scaling down microfluidic droplet possible: fluorophore leakage-free report 3 orders magnitude-improved sensitivity compared absorbance-based systems, resolution 361,000 product molecules per droplet. Our approach enables nonfluorogenic droplet-based enrichments, applicability hydrolases imine reductases (IREDs). To demonstrate assay's readiness combinatorial experiments, one round was performed select glycosidase processing natural substrate, beechwood xylan, improved kinetic parameters pool >106 mutagenized sequences.

Language: Английский

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Artificial metalloenzymes

Nature Reviews Methods Primers, Journal Year: 2024, Volume and Issue: 4(1)

Published: Nov. 1, 2024

Language: Английский

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