Challenges and Future Perspectives in Photocatalysis: Conclusions from an Interdisciplinary Workshop.

Photocatalysis is a versatile and rapidly developing field with applications spanning artificial photosynthesis, photo-biocatalysis, photoredox catalysis in solution or supramolecular structures, utilization of abundant metals and organocatalysts, sustainable synthesis, and plastic degradation. In this Perspective, we summarize conclusions from an interdisciplinary workshop of young principal investigators held at the Lorentz Center in Leiden in March 2023. We explore how diverse fields within photocatalysis can benefit from one another.

A De Novo Metalloenzyme for Cerium Photoredox Catalysis.

Cerium photoredox catalysis has emerged as a powerful strategy to activate molecules under mild conditions. Radical intermediates are formed using visible light and simple complexes of the earth-abundant lanthanide. Here, we report an artificial photoenzyme enabling this chemistry inside a protein.

Minor Actinides Can Replace Essential Lanthanides in Bacterial Life.

Certain f-block elements-the lanthanides-have biological relevance in the context of methylotrophic bacteria. The respective strains incorporate these 4 f elements into the active site of one of their key metabolic enzymes, a lanthanide-dependent methanol dehydrogenase. In this study, we investigated whether actinides, the radioactive 5 f elements, can replace the essential 4 f elements in lanthanide-dependent bacterial metabolism.

Americium preferred: lanmodulin, a natural lanthanide-binding protein favors an actinide over lanthanides.

The separation and recycling of lanthanides is an active area of research with a growing demand that calls for more environmentally friendly lanthanide sources. Likewise, the efficient and industrial separation of lanthanides from the minor actinides (Np, Am-Fm) is one of the key questions for closing the nuclear fuel cycle; reducing costs and increasing safety. With the advent of the field of lanthanide-dependent bacterial metabolism, bio-inspired applications are in reach.

Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase.

The roles of local interactions in the laboratory evolution of a highly active, computationally designed retroaldolase (RA) are examined. Partial Order Optimum Likelihood (POOL) is used to identify catalytically important amino acid interactions in several RA95 enzyme variants. The series RA95.

Design and engineering of artificial metalloproteins: from de novo metal coordination to catalysis.

Metalloproteins are essential to sustain life. Natural evolution optimized them for intricate structural, regulatory and catalytic functions that cannot be fulfilled by either a protein or a metal ion alone. In order to understand this synergy and the complex design principles behind the natural systems, simpler mimics were engineered from the bottom up by installing de novo metal sites in either natural or fully designed, artificial protein scaffolds.

Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion.

De novo protein design has succeeded in generating a large variety of globular proteins, but the construction of protein scaffolds with cavities that could accommodate large signaling molecules, cofactors, and substrates remains an outstanding challenge. The long, often flexible loops that form such cavities in many natural proteins are difficult to precisely program and thus challenging for computational protein design. Here we describe an alternative approach to this problem.

Lysine acylation using conjugating enzymes for site-specific modification and ubiquitination of recombinant proteins.

Enzymes are powerful tools for protein labelling due to their specificity and mild reaction conditions. Many protocols, however, are restricted to modifications at protein termini, rely on non-peptidic metabolites or require large recognition domains. Here we report a chemoenzymatic method, which we call lysine acylation using conjugating enzymes (LACE), to site-specifically modify folded proteins at internal lysine residues.

Emergence of a Negative Activation Heat Capacity during Evolution of a Designed Enzyme.

Temperature influences the reaction kinetics and evolvability of all enzymes. To understand how evolution shapes the thermodynamic drivers of catalysis, we optimized the modest activity of a computationally designed enzyme for an elementary proton-transfer reaction by nearly 4 orders of magnitude over 9 rounds of mutagenesis and screening. As theorized for primordial enzymes, the catalytic effects of the original design were almost entirely enthalpic in origin, as were the rate enhancements achieved by laboratory evolution.

Directed Evolution of Protein Catalysts.

Directed evolution is a powerful technique for generating tailor-made enzymes for a wide range of biocatalytic applications. Following the principles of natural evolution, iterative cycles of mutagenesis and screening or selection are applied to modify protein properties, enhance catalytic activities, or develop completely new protein catalysts for non-natural chemical transformations. This review briefly surveys the experimental methods used to generate genetic diversity and screen or select for improved enzyme variants.

Bap (Sil1) regulates the molecular chaperone BiP by coupling release of nucleotide and substrate.

BiP is the endoplasmic member of the Hsp70 family. BiP is regulated by several co-chaperones including the nucleotide-exchange factor (NEF) Bap (Sil1 in yeast). Bap is a two-domain protein.

Optimization of Enzyme Mechanism along the Evolutionary Trajectory of a Computationally Designed (Retro-)Aldolase.

De novo biocatalysts have been successfully generated by computational design and subsequent experimental optimization. Here, we examined the evolutionary history of the computationally designed (retro-)aldolase RA95. The modest activity of the starting enzyme was previously improved 10-fold over many rounds of mutagenesis and screening to afford a proficient biocatalyst for enantioselective cleavage and synthesis of β-hydroxyketones.

Efficient laboratory evolution of computationally designed enzymes with low starting activities using fluorescence-activated droplet sorting.

De novo biocatalysts with non-natural functionality are accessible by computational enzyme design. The catalytic activities obtained for the initial designs are usually low, but can be optimized significantly by directed evolution. Nevertheless, rate accelerations approaching the level of natural enzymes can only be achieved over many rounds of tedious and time-consuming laboratory evolution.

Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction.

States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods. In addition to apo and ligand-bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side-chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions.

trans-Acting arginine residues in the AAA+ chaperone ClpB allosterically regulate the activity through inter- and intradomain communication.

The molecular chaperone ClpB/Hsp104, a member of the AAA+ superfamily (ATPases associated with various cellular activities), rescues proteins from the aggregated state in collaboration with the DnaK/Hsp70 chaperone system. ClpB/Hsp104 forms a hexameric, ring-shaped complex that functions as a tightly regulated, ATP-powered molecular disaggregation machine. Highly conserved and essential arginine residues, often called arginine fingers, are located at the subunit interfaces of the complex, which also harbor the catalytic sites.

RNA specificity and regulation of catalysis in the eukaryotic polynucleotide kinase Clp1.

RNA-specific polynucleotide kinases of the Clp1 subfamily are key components of various RNA maturation pathways. However, the structural basis explaining their substrate specificity and the enzymatic mechanism is elusive. Here, we report crystal structures of Clp1 from Caenorhabditis elegans (ceClp1) in a number of nucleotide- and RNA-bound states along the reaction pathway.

Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor.

ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically.

The molecular mechanism of Hsp100 chaperone inhibition by the prion curing agent guanidinium chloride.

The Hsp100 chaperones ClpB and Hsp104 utilize the energy from ATP hydrolysis to reactivate aggregated proteins in concert with the DnaK/Hsp70 chaperone system, thereby playing an important role in protein quality control. They belong to the family of AAA+ proteins (ATPases associated with various cellular activities), possess two nucleotide binding domains per monomer (NBD1 and NBD2), and oligomerize into hexameric ring complexes. Furthermore, Hsp104 is involved in yeast prion propagation and inheritance.

Coupling of oligomerization and nucleotide binding in the AAA+ chaperone ClpB.

Members of the family of ATPases associated with various cellular activities (AAA+) typically form homohexameric ring complexes and are able to remodel their substrates, such as misfolded proteins or protein-protein complexes, in an ATP-driven process. The molecular mechanism by which ATP hydrolysis is coordinated within the multimeric complex and the energy is converted into molecular motions, however, is poorly understood. This is partly due to the fact that the oligomers formed by AAA+ proteins represent a highly complex system and analysis depends on simplification and prior knowledge.

A sterile alpha-motif domain in NafY targets apo-NifDK for iron-molybdenum cofactor delivery via a tethered domain.

NafY participates in the final steps of nitrogenase maturation, having a dual role as iron-molybdenum cofactor (FeMo-co) carrier and as chaperone to the FeMo-co-deficient apo-NifDK (apo-dinitrogenase). NafY contains an N-terminal domain of unknown function (n-NafY) and a C-terminal domain (core-NafY) necessary for FeMo-co binding. We show here that n-NafY and core-NafY have very weak interactions in intact NafY.