R.B. Leveson-Gower

Nature Communications, Published online: 13 July 2023; doi:10.1038/s41467-023-39819-1

Viral occlusion bodies are robust protein crystals that encapsulate virions of some insect viruses. Here, the authors determine the nudivirus occlusion body structure and describe common principles of occlusion body structure.

Nature Chemical Biology, Published online: 13 July 2023; doi:10.1038/s41589-023-01387-2

Tian et al. developed a bacterial orthogonal DNA replication system by harnessing the temperate phage GIL16 DNA replication machinery, which provides a powerful tool for continuous evolution in prokaryotic cells.

“My most important contribution to open science is perhaps our ongoing work advocating for the use of scientific color maps … I advise my students to expect that an experiment might take at least three attempts: One to fail and learn from. One to improve and get a grip on things. One to get it halfway right and acquire meaningful data.” Find out more about Felix Kaspar in his Introducing … Profile.

Nature, Published online: 05 July 2023; doi:10.1038/s41586-023-06288-x

An engineered minimal cell evolves to escape the negative consequences of genome streamlining.

Selective, one-step C-H activation of fatty acids from biomass is an attractive concept in sustainable chemistry. Biocatalysis has shown promise for generating high-value hydroxy acids but to date enzyme discovery has relied on laborious screening and produced limited hits, which predominantly oxidise the sub-terminal positions of fatty acids. Here we show that ancestral sequence reconstruction (ASR) is an effective tool to explore the sequence-activity landscape of a family of multi-domain, self-sufficient P450 monooxygenases. We resurrected eleven catalytically active CYP116B ancestors, each with a unique regioselectivity fingerprint that varied from sub-terminal in the older ancestors to mid-chain in the lineage leading to the extant, P450-TT. In lineages leading to extant enzymes in thermophiles, thermostability increased from ancestral to extant forms, as expected if thermophily had arisen de novo. Our studies show that ASR can be applied to multi-domain enzymes to develop active, self- sufficient monooxygenases as regioselective biocatalysts for fatty acid hydroxylation.

8-Azido-3,8-dideoxy-α/β-ᴅ-manno-oct-2-ulosonic acid (Kdo-8-N3) is a Kdo derivative used in metabolic labeling of lipopolysaccharide (LPS) structures found on the cell membrane of Gram-negative bacteria. Several studies have reported successful labeling of LPS using Kdo-8-N3 and visualization of LPS by a fluorescent reagent through click chemistry on a selection of Gram-negative bacteria such as Escherichia coli strains, Salmonella typhimurium, and Myxococcus xanthus. Motivated by the large promise of Kdo-8-N3 to be useful in the investigation of LPS biosynthesis and cell surface labeling across different strains, we set out to explore the variability in nature and efficiency of LPS labeling using Kdo-8-N3 in a variety of E. coli strains and serotypes. We optimized the chemical synthesis of Kdo-8-N3 and subsequently used Kdo-8-N3 to metabolically label pathogenic E. coli strains from commercial and clinical origin. Interestingly, different extents of labeling were observed in different E. coli strains, which seemed to be dependent also on growth media, and the majority of labeled LPS appears to be of the ‘rough’ LPS variant, as visualized using SDS-PAGE and fluorescence microscopy. This knowledge is important for future application of Kdo-8-N3 in the study of LPS biosynthesis and dynamics, especially when working with clinical isolates.

Protein–protein interactions (PPIs) are intriguing targets in drug discovery and development. Peptides are well suited to target PPIs, which typically present with large surface areas lacking distinct features and deep binding pockets. To improve binding interactions to these topologies by PPI-focused therapeutics and advance their development, potential ligands can be equipped with electrophilic groups to enable binding through covalent mechanisms of action. We report a strategy termed electrophile scanning to identify reactivity hotspots in a known peptide ligand. Cysteine mutants of the ligand are used to install protein-reactive modifiers via a palladium oxidative addition complex (Pd-OAC). Reactivity hotspots are revealed by cross-linking reactions with the target protein under physiological conditions. In a system with the 9-mer peptide antigen VL9 and MHC class I receptor HLA-E, we identify two reactivity hotspots that afford up to 87% conversion to the protein–peptide conjugate within 4 hours. The reactions are specific to the target protein in vitro and dependent on the peptide sequence. Moreover, the cross-linked peptide successfully inhibits molecular recognition of HLA-E by CD94─NKG2A possibly due to structural changes enacted at the PPI interface. The results illustrate the potential of electrophile scanning as a tool for rapid discovery and development of covalent peptide binders.

Directed evolution facilitates enzyme engineering via iterative rounds of mutagenesis. Despite the wide applications of high-throughput screening, building “smart libraries” to effectively identify beneficial variants remains a major challenge in the community. Here, we developed a new computational directed evolution protocol based on EnzyHTP, a software we have previously reported to automate enzyme modeling. To enhance the throughput efficiency, we implemented an adaptive resource allocation strategy that dynamically allocates different types of computing resources (e.g., GPU/CPU) based on the specific need of an enzyme modeling sub-task in the workflow. We implemented the strategy as a Python library and tested the library using fluoroacetate dehalogenase as a model enzyme. The results show that comparing to fixed resource allocation where both CPU and GPU are on-call for use during the entire workflow, applying adaptive resource allocation can save 87% CPU hours and 14% GPU hours. Furthermore, we constructed a computational directed evolution protocol under the framework of adaptive resource allocation. The workflow was tested against two rounds of mutational screening in the directed evolution experiments of Kemp eliminase with a total of 184 mutants. Using folding stability and electrostatic stabilization energy as computational readout, we reproduced three out of the four experimentally-observed target variants. Enabled by the workflow, the entire computation task (i.e., 18.4 μs MD and 18,400 QM single point calculations) completes in three days of wall clock time using ~30 GPUs and ~1000 CPUs.

Nature Chemistry, Published online: 19 June 2023; doi:10.1038/s41557-023-01262-6

Protein translation is the ultimate paradigm for sequence-defined polymer synthesis. To introduce non-canonical monomers into the genetic code of living organisms, pairs of biomolecules known as aminoacyl-tRNA synthetases (aaRSs) and transfer RNAs (tRNAs) are required. The discovery and engineering of five such pairs, that do not interfere with each other or the aaRS–tRNA pairs of a bacterial host, sets the stage for highly modified genetically encoded biopolymers.

We report on a readily available indole thiolate organocatalyst that, upon excitation with light, can activate via single electron reduction a wide variety of typically inert electron-rich substrates, including strong C−F, C−Cl, and C−O bonds in both aromatic and aliphatic substrates. The protocol is also useful for the borylation and phosphorylation of inert substrates, exhibiting high functional group tolerance.

Abstract

Due to their strong covalent bonds and low reduction potentials, activating inert substrates is challenging. Recent advances in photoredox catalysis offered a number of solutions, each of which useful for activating specific inert bonds. Developing a general catalytic platform that can consistently target a broad range of inert substrates would be synthetically useful. Herein, we report a readily available indole thiolate organocatalyst that, upon excitation with 405 nm light, acquires a strongly reducing power. This excited-state reactivity served to activate, by single-electron reduction, strong C−F, C−Cl, and C−O bonds in both aromatic and aliphatic substrates. This catalytic platform was versatile enough to promote the reduction of generally recalcitrant electron-rich substrates (E_red<−3.0 V vs SCE), including arenes that afforded 1,4-cyclohexadienes. The protocol was also useful for the borylation and phosphorylation of inert substrates with a high functional group tolerance. Mechanistic studies identified an excited-state thiolate anion as responsible of the highly reducing reactivity.

Protein tyrosine phosphatases are crucial regulators of cellular signaling. Their activity is regulated by the motion of a conserved loop, the WPD-loop, from a catalytically inactive open to a catalytically active closed conformation. WPD-loop motion optimally positions a catalytically critical residue into the active site, and is directly linked to the turnover number of these enzymes. Crystal structures of chimeric PTPs constructed by grafting parts of the WPD-loop sequence of PTP1B onto the scaffold of YopH showed WPD-loops in a wide-open conformation never previously observed in either parent enzyme. This wide-open conformation has, however, been observed upon binding of small molecule inhibitors to other PTPs, suggesting the potential of targeting it for drug discovery efforts. Here, we have performed simulations of both enzymes and show that there are negligible energetic differences in the chemical step of catalysis, but significant differences in the dynamical properties of the WPD-loop. Detailed interaction network analysis provides insight into the molecular basis for this population shift to a wide-open conformation. Taken together, our study provides insight into the links between loop dynamics and chemistry in these YopH variants specifically, and how WPD-loop dynamic can be engineered through modification of the internal protein interaction network.

Science, Volume 380, Issue 6650, Page 1150-1154, June 2023.

Science, Volume 380, Issue 6650, Page 1188-1192, June 2023.

Diels-Alderases perform an essential step in the biosynthesis of bioactive spirotetronates. To expand the understanding of such enzymes a cyclase library was created, which made it possible to identify a novel spirotetronate cyclase from a metagenome mining approach. Structural elucidation of both the enzyme by X-ray crystallography and the product by NMR helped us to gain further insights into the essential features of how these enzymes perform complex cyclisations.

Abstract

Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a [4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved [4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted [4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases.

Enzymes can offer an enticing tool for building complex chemical scaffolds through succinct routes and under mild conditions. Yet, the common application of biocatalysts in organic synthesis is often hampered by unpredictable substrate scope and scalability challenges, deterring the planning of biocatalytic steps at the retrosynthetic planning stage. Herein, we detail a method using a sequence similarity network to curate a library of non-heme iron (NHI)-dependent enzymes capable of performing complexity generating biocatalytic transformations. In the course of this study, we probed the substrate scope of TropC-like enzymes to furnish a range of beta-hydroxytropolone products. The potential to access diverse scaffolds was investigated and a variety of tropolone-containing molecules were prepared on milligram-scale. Furthermore, chemoenzymatically generated tropolones were transformed through a variety of chemistries to achieve the total synthesis of stipitaldehyde, an abbreviated formal synthesis of deoxyepolone B, and additional tropolone building blocks with a high density of functional handles. This work lays the foundation for using NHI enzymes in retrosynthetic planning of complex molecules and natural product analogues.

Nature Chemistry, Published online: 12 June 2023; doi:10.1038/s41557-023-01235-9

The metal-dependent, bifunctional isoprenyl diphosphate synthase PcIDS1 from the leaf beetle Phaedon cochleariae integrates substrate, product and metal-ion concentrations to tune its dynamic reactivity. Now structural and functional analyses reveal that this enzyme uses both catalytic centres to form geranyl pyrophosphate, while one domain is inactivated during farnesyl pyrophosphate production.

Nature Catalysis, Published online: 08 June 2023; doi:10.1038/s41929-023-00963-y

Pyridoxal 5′-phosphate (PLP)-dependent enzymes that catalyse Mannich reactions were unknown. Now, it is reported that the PLP-dependent enzyme LolT catalyses a 5-endo-trig Mannich cyclization reaction during the pyrrolizidine core scaffold formation in loline biosynthesis, and its crystal structure is solved.