Fabrizio

Nature, Published online: 08 May 2024; doi:10.1038/s41586-024-07391-3

A completely genetically encoded boronic-acid-containing designer enzyme was created and characterized using X-ray crystallography, high-resolution mass spectrometry and 11B NMR spectroscopy, allowing chemistry that is unknown in nature and currently not possible with small-molecule catalysts.

Nature Communications, Published online: 20 March 2025; doi:10.1038/s41467-025-58038-4

Enzyme kinetic parameter prediction is a challenge in enzyme discovery and engineering. Here, the authors train a robust deep learning model CataPro to predict enzyme kinetic parameters and validate its practicality through wet-lab experiments.

An efficient artificial copper-dependent Michaelase featuring a metal-binding unnatural amino acid, e. g. bipyridyl alanine (BpyA), was optimized through directed evolution and applied in catalytic asymmetric additions of 2-acetyl azaarenes to nitroalkenes. Various chiral γ-nitro butyric acid derivatives were obtained in good yields with high enantioselectivities. Moreover, the reaction was performed at preparative scale and the product was used in follow-up derivatization reactions towards various high-value-added pharmaceutically relevant compounds.

Abstract

Artificial metalloenzymes (ArMs) are an attractive approach to achieving “new to nature” biocatalytic transformations. In this work, a novel copper-dependent artificial Michaelase (Cu_Michaelase) comprising a genetically encoded copper-binding ligand, i. e. (2,2-bipyridin-5-yl)alanine (BpyA), was developed. For the first time, such an ArM containing a non-canonical metal-binding amino acid was successfully optimized through directed evolution. The evolved Cu_Michaelase was applied in the copper-catalyzed asymmetric addition of 2-acetyl azaarenes to nitroalkenes, yielding various γ-nitro butyric acid derivatives, which are precursors for a range of high-value-added pharmaceutically relevant compounds, with good yields and high enantioselectivities (up to >99 % yield and 99 % ee). Additionally, the evolved variant could be further used in a preparative-scale synthesis, providing chiral products for diverse derivatizations. X-ray crystal structure analysis confirmed the binding of Cu(II) ions to the BpyA residues and showed that, in principle, there is sufficient space for the 2-acetyl azaarene substrate to coordinate. Kinetic studies showed that the increased catalytic efficiency of the evolved enzyme is due to improvements in apparent K _M for both substrates and a notable threefold increase in apparent k _cat for 2-acetyl pyridine. This work illustrates the potential of artificial metalloenzymes exploiting non-canonical metal-binding ligands for new-to-nature biocatalysis.

Promiscuity guided evolution of the decarboxylative aldolase, UstD, resulted in an efficient biocatalyst for synthesis of tertiary γ-hydroxy non-canonical amino acids. Simultaneous collection of variant activity and promiscuity was enabled by competition screening during directed evolution. Changes in promiscuity effectively identified distal residues that influence catalysis, a longstanding challenge in protein engineering.

Abstract

Many applications of enzymes benefit from activity on structurally diverse substrates. Here, we sought to engineer the decarboxylative aldolase UstD to perform a challenging C−C bond forming reaction with ketone electrophiles. The parent enzyme had only low levels of activity, portending multiple rounds of directed evolution and a possibility that mutations may inadvertently increase the specificity of the enzyme for a single model screening substrate. We show how to intentionally guide UstD towards generality through multi-generational directed evolution using substrate-multiplexed screening (SUMS). Mutations outside of the active site that impact catalytic function were immediately revealed by shifts in promiscuity, even when the overall activity was lower. By re-targeting these distal residues that couple to the active site with saturation mutagenesis, broadly activating mutations were readily identified. When analyzing active site mutants, SUMS identified both specialist enzymes that would have more limited utility as well as generalist enzymes with complementary activity on diverse substrates. These new UstD enzymes catalyze convergent synthesis of non-canonical amino acids bearing tertiary alcohol side chains. This methodology is easy to implement and enables the rapid and effective evolution of enzymes to catalyze desirable new functions.

Amino-acid residues distant from an enzymes active site are known to influence catalysis, but their mechanistic contributions to the catalytic cycle remain poorly understood. Here, we investigate the structural, functional, and mechanistic impacts of distal and active-site mutations discovered through directed evolution of the computationally designed retro-aldolase RA95. Active-site mutations improve catalytic efficiency by 3,600-fold, while distal mutations alone offer no improvement. When combined with active-site mutations, distal mutations further increase efficiency by 6-fold, demonstrating an epistatic effect. X-ray crystallography and molecular dynamics simulations reveal that distal mutations promote active site opening by altering loop dynamics. Kinetic solvent viscosity effects and electrostatic analysis show that distal mutations accelerate the chemical transformation by 100-fold, shifting the rate-limiting step to product release, which is further accelerated by the increased opening of the active site. These findings highlight the critical role of distal residues in shaping the active-site environment and facilitating the structural dynamics essential for progression through the catalytic cycle.

Chemical modifications based on dehydroalanine (Dha) have attracted considerable attention in the field of peptide and protein functionalization due to their high efficiency and site selectivity. This review summarizes the latest progress on the Dha-specific modification and diversification of peptides and proteins, as well as their applications in chemical biology and drug discovery.

In recent years, chemical modification techniques based on dehydroalanine (Dha) have become a prominent area in the functionalization of peptides and proteins due to their high efficiency and site selectivity. This article reviews the recent advancements in the modification of Dha-containing peptides and proteins. Focusing on the chemical properties of Dha, the advantages of nucleophilic addition reactions, free radical chemistry, metal-catalyzed cross-coupling reactions, and cycloaddition reactions in peptide and protein modification are discussed, as well as their applicability in the development of novel biocompatible methods. Additionally, the article explores the current limitations of these techniques and highlights future challenges that need to be addressed.

At the intersection of chemical biology, plant imaging, and contemporary art, this review introduces the concept of chembioart. By tracing how chemical reporters illuminate plant biomolecules in vivo, their role not only in advancing scientific understanding but also in inspiring transdisciplinary collaborations and visual expression is highlighted.

Chemical biology has reshaped the ability to investigate complex biological systems at the molecular level. In this context, chemical reporters have become important tools for labeling and tracking biomolecules in living systems with spatial and temporal precision. In plant biology, they provide an alternative to genetic approaches and allow the study of dynamic processes in species or organs that are not easily accessible. Through the use of click and bioorthogonal chemistry, small-molecule probes can be metabolically incorporated into specific molecular scaffolds such as sugars, monolignols, amino acids, and lipids. These probes make it possible to follow events like glycosylation, lignification, lipid turnover, or protein synthesis in living plant tissues. This review presents an overview of current chemical reporter strategies, from molecular design and synthetic considerations to their application in plant imaging. Herein, how these tools have contributed to the development of plant chemical biology by enabling precise and modular investigations of plant structure and metabolism is described. Herein, it is also examined how chemical reporters have entered interdisciplinary contexts, including collaborations between science and the arts. By converting molecular-level information into visual and sensory formats, these approaches open new perspectives for research, education, and communication across scientific and creative disciplines.

Nature Catalysis, Published online: 03 November 2025; doi:10.1038/s41929-025-01436-0

The creation of artificial metalloenzymes compatible with complex biological settings could enable broad applications. Now a de novo-designed artificial metalloenzyme containing an abiological ruthenium cofactor is reported and optimized for ring-closing metathesis in the cytoplasm of whole cells.

The Nobel Prize for Chemistry 2024 was jointly awarded to David Baker for computational protein design and to Demis Hassabis and John Jumper for protein structure prediction. This highlight showcases the impact of the Nobel prize laureates’ contributions and summarizes the history, state of the art, applications and future directions of these methods.

Abstract

The ability to predict and design protein structures has led to numerous applications in medicine, diagnostics and sustainable chemical manufacture. In addition, the wealth of predicted protein structures has advanced our understanding of how life's molecules function and interact. Honouring the work that has fundamentally changed the way scientists research and engineer proteins, the Nobel Prize in Chemistry in 2024 was awarded to David Baker for computational protein design and jointly to Demis Hassabis and John Jumper, who developed AlphaFold for machine-learning-based protein structure prediction. Here, we highlight notable contributions to the development of these computational tools and their importance for the design of functional proteins that are applied in organic synthesis. Notably, both technologies have the potential to impact drug discovery as any therapeutic protein target can now be modelled, allowing the de novo design of peptide binders and the identification of small molecule ligands through in silico docking of large compound libraries. Looking ahead, we highlight future research directions in protein engineering, medicinal chemistry and material design that are enabled by this transformative shift in protein science.

Nature Biotechnology, Published online: 23 April 2024; doi:10.1038/s41587-024-02214-2

Metrics that predict the success of folding and the activity of designed protein sequences are developed and experimentally validated.

Nature Catalysis, Published online: 23 April 2024; doi:10.1038/s41929-024-01137-0

Synthetic methylotrophic organisms provide potential for valorization of greenhouse gas-derived methanol. Here an Escherichia coli strain is generated that reaches a similar growth rate on methanol to many natural methylotrophs and is capable of producing chemicals from this carbon source.

Publication date: 28 May 2024

Source: Cell Reports, Volume 43, Issue 5

Author(s): Guang Yang, Ognjen Pećanac, Hein J. Wijma, Henriëtte J. Rozeboom, Gonzalo de Gonzalo, Marco W. Fraaije, Maria Laura Mascotti

S-Adenosylmethionine assumes a crucial role in biocatalytic alkylation. To improve the catalytic ability of BxHMT towards methyl toluene sulfonate, a strategy named dynamic cross-correlation network analysis (DCCNA) was proposed for the identification of potential mutation sites, both distal and proximal. Experimental results proved the efficiency of the approach, which is expected to be a useful tool for other enzyme engineering studies.

Abstract

Halide methyltransferases (HMTs) provide an effective way to regenerate S-adenosyl methionine (SAM) from S-adenosyl homocysteine and reactive electrophiles, such as methyl iodide (MeI) and methyl toluene sulfonate (MeOTs). As compared with MeI, the cost-effective unnatural substrate MeOTs can be accessed directly from cheap and abundant alcohols, but shows only limited reactivity in SAM production. In this study, we developed a dynamic cross-correlation network analysis (DCCNA) strategy for quickly identifying hot spots influencing the catalytic efficiency of the enzyme, and applied it to the evolution of HMT from Paraburkholderia xenovorans. Finally, the optimal mutant, M4 (V55T/C125S/L127T/L129P), exhibited remarkable improvement, with a specific activity of 4.08 U/mg towards MeOTs, representing an 82-fold increase as compared to the wild-type (WT) enzyme. Notably, M4 also demonstrated a positive impact on the catalytic ability with other methyl donors. The structural mechanism behind the enhanced enzyme activity was uncovered by molecular dynamics simulations. Our work not only contributes a promising biocatalyst for the regeneration of SAM, but also offers a strategy for efficient enzyme engineering.

Multiple enzymes catalyze the formation of pyrroloindoles from indoles, usually coupled with a functional group transfer in the 3-position. In this work, two high-throughput complementary absorbance-based assays were developed for the monitoring of substrate depletion (indole) and product formation (pyrroloindole). The assays were used successfully for enzymatic activity determination, but can be also used for the quantification of natural products.

Abstract

Indoles and pyrroloindoles are structural motifs present in many biologically active natural products. Multiple classes of enzymes catalyze the transformation of indoles into pyrroloindoles via group transfer followed by intramolecular cyclization, such as peroxydases, methyltransferases, and prenyltransferases. Due to the selective introduction of a stereogenic center, these enzymes receive increasing attention as catalytic tools for the production of pharmacologically relevant compounds. Two new colorimetric assays are described in this work, which allow for the quantification of such enzymatic reactions from the perspective of the substrate and the product. For the substrates, the indole assay is based on a modified version of the Ehrlich test, with the use of light as a driving force for color formation. The pyrroloindole assay uses cerium sulfate as a reagent for the colorimetric quantification of the enzymatic products. The assays are complementary and both were successfully utilized for enzymatic activity determination of a C3-indole methyltransferase. They can facilitate high-throughput screening of mutant libraries, offering support for the engineering of such enzymes, but can also be used as stand-alone methods for the detection and quantification of natural products.

Proteins and enzymes can be repurposed by the introduction of artificial cofactors or non-canonical amino acids (ncAAs). These artificial biocatalytic constructs turned into valuable tools to perform new-to-nature reactions with biocatalysts increasing their scope. This perspective focuses on the limitations and future application for in vivo biosynthetic pathways.

Abstract

Astonishing progress has been achieved in unlocking new-to-nature biocatalysis in the past decades. The progress in protein engineering enabled research to efficiently incorporate artificial structural elements into enzyme design. Recent trends include cofactor mimetics, artificial metalloenzymes and non-canonical amino acids. In this perspective article, we present the state-of-the-art, discuss recent examples and our view on what we call artificial biocatalysis. Although these artificial systems undoubtedly increase the scope of biocatalysis, their applicability remains challenging. Fundamental questions regarding the impact of this research field are addressed in this perspective.

An M9 minimum media based on ¹³C-depleted glucose and ¹⁵N-depleted salt dissolved in D,¹⁸O-depleted water (Depleted media) was formulated. E. coli bacteria grow faster in Depleted media compared with isotopically natural media (Normal media). In addition, four different enzymes recombinantly produced in Depleted media showed faster kinetics compared with the enzymes produced in Normal media.

Abstract

Inorganic materials depleted of heavy stable isotopes are known to deviate strongly in some physicochemical properties from their isotopically natural counterparts. Here we explored for the first time the effect of simultaneous depletion of the heavy carbon, hydrogen, oxygen and nitrogen isotopes on the bacterium E. coli and the enzymes expressed in it. Bacteria showed faster growth, with most proteins exhibiting higher thermal stability, while for recombinant enzymes expressed in depleted media, faster kinetics was discovered. At room temperature, luciferase, thioredoxin and dihydrofolate reductase and Pfu DNA polymerase showed up to a 250 % increase in activity compared to the native counterparts, with an additional ∼50 % increase at 10 °C. Diminished conformational and vibrational entropy is hypothesized to be the cause of the accelerated kinetics. Ultralight enzymes may find an application where extreme reaction rates are required.

Nature Chemical Biology, Published online: 20 November 2023; doi:10.1038/s41589-023-01484-2

Oxygen sensitivity hampers applications of metal-dependent CO2 reductases. Here, Oliveira et al. describe how an allosteric disulfide bond controls the activity of a CO2 reductase, preventing its physiological reduction during transient O2 exposure and allowing aerobic handling of the enzyme.

Abstract

Synthetic binding proteins have emerged as modulators of protein functions through protein–protein interactions (PPIs). Because PPIs are influenced by the structural dynamics of targeted proteins, investigating whether the synthetic-binders-based strategy is applicable for proteins with large conformational changes is important. This study demonstrates the applicability of monobodies (fibronectin type-III domain-based synthetic binding proteins) in regulating the functions of proteins that undergo tens-of-angstroms-scale conformational changes, using an example of the A55C/C77S/V169C triple mutant (Adk_tm; a phosphoryl transfer-catalyzing enzyme with a conformational change between OPEN/CLOSED forms). Phage display successfully developed monobodies that recognize the OPEN form (substrate-unbound form), but not the CLOSED form of Adk_tm. Two OPEN form-specific clones (OP-2 and OP-4) inhibited Adk_tm kinase activity. Epitope mapping with a yeast-surface display/flow cytometry indicated that OP-2 binds to the substrate-entry side of Adk_tm, whereas OP-4 binding occurs at another site. Small angle X-ray scattering  coupled with size-exclusion chromatography (SEC-SAXS) indicated that OP-4 binds to the hinge side opposite to the substrate-binding site of Adk_tm, retaining the whole OPEN-form structure of Adk_tm. Titration of the OP-4–Adk_tm complex with Ap₅A, a transition-state analog of Adk_tm, showed that the conformational shift to the CLOSED form was suppressed although Adk_tm retained the OPEN-form (i.e., substrate-binding ready form). These results show that OP-4 captures and stabilizes the OPEN-form state, thereby affecting the hinge motion. These experimental results indicate that monobody-based modulators can regulate the functions of proteins that show tens-of-angstroms-scale conformational changes, by trapping specific conformational states generated during large conformational change process that is essential for function exertion.

An aqueous Pictet-Spengler annulation has been interfaced with whole-cell alcohol oxidation. This one-pot cascade reaction converts aliphatic alcohols and tyramines or tryptamines into alkaloid heterocycles under mild, aqueous conditions, delivering tetrahydroisouinolines and tryptolines in >90 % and >40 % isolated yield, respectively, with excellent regioselectivity.

Abstract

Biocatalytic processes are highly selective and specific. However, their utility is limited by the comparatively narrow scope of enzyme-catalysed transformations. To expand product scope, we are developing biocompatible processes that combine biocatalytic reactions with chemo-catalysis in single-flask processes. Here, we show that a chemocatalysed Pictet-Spengler annulation can be interfaced with biocatalysed alcohol oxidation. This two-step, one-pot cascade reaction converts tyramine and aliphatic alcohols to tetrahydroisoquinoline alkaloids in aqueous buffer at mild pH. Tryptamine derivatives are also efficiently converted to tryptolines. Optimization of stoichiometry, pH, reaction time, and whole-cell catalyst deliver the tetrahydroisouinolines and tryptolines in >90 % and >40 % isolated yield, respectively, with excellent regioselectivity.

Abstract

G protein-coupled receptors (GPCRs) are medically important membrane proteins that sample inactive, intermediate, and active conformational states characterized by relatively slow interconversions (~μs–ms). On a faster timescale (~ps–ns), the conformational landscape of GPCRs is governed by the rapid dynamics of amino acid side chains. Such dynamics are essential for protein functions such as ligand recognition and allostery. Unfortunately, technical challenges have almost entirely precluded the study of side-chain dynamics for GPCRs. Here, we investigate the rapid side-chain dynamics of a thermostabilized α_1B-adrenergic receptor (α_1B-AR) as probed by methyl relaxation. We determined order parameters for Ile, Leu, and Val methyl groups in the presence of inverse agonists that bind orthosterically (prazosin, tamsulosin) or allosterically (conopeptide ρ-TIA). Despite the differences in the ligands, the receptor's overall side-chain dynamics are very similar, including those of the apo form. However, ρ-TIA increases the flexibility of Ile176^4×56 and possibly of Ile214^5×49, adjacent to Pro215^5×50 of the highly conserved P^5×50I^3×40F^6×44 motif crucial for receptor activation, suggesting differences in the mechanisms for orthosteric and allosteric receptor inactivation. Overall, increased Ile side-chain rigidity was found for residues closer to the center of the membrane bilayer, correlating with denser packing and lower protein surface exposure. In contrast to two microbial membrane proteins, in α_1B-AR Leu exhibited higher flexibility than Ile side chains on average, correlating with the presence of Leu in less densely packed areas and with higher protein-surface exposure than Ile. Our findings demonstrate the feasibility of studying receptor-wide side-chain dynamics in GPCRs to gain functional insights.