Towards controlling activity of a peptide asparaginyl ligase (PAL) by lumazine synthetase compartmentalization.

Peptide asparaginyl ligases (PALs) hold significant potential in protein bioconjugation due to their excellent kinetic properties and broad substrate compatibility. However, realizing their full potential in biocatalytic applications requires precise control of their activity. Inspired by nature, we aimed to compartmentalize a representative PAL, OaAEP1-C247A, within protein containers to create artificial organelles with substrate sorting capability. Two encapsulation approaches were explored using engineered lumazine synthases (AaLS). The initial strategy involved tagging the PAL with a super-positively charged GFP(+36) for encapsulation into the super-negatively charged AaLS-13 variant, but it resulted in undesired truncation of the enzyme. The second approach involved genetic fusion of the OaAEP1-C247A with a circularly permutated AaLS variant (cpAaLS) and its co-production with AaLS-13, which successfully enabled compartmentalization of the PAL within a patch-work protein cage. Although the caged PAL retained its activity, it was significantly reduced compared to the free enzyme (∼30-40-fold), likely caused by issues related to OaAEP1-C247A stability and folding. Nevertheless, these findings demonstrated the feasibility of the AaLS encapsulation approach and encourage further optimization in the design of peptide-ligating artificial organelles in E. coli, aiming for a more effective and stable system for protein modifications.

Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion.

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D-proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR-based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts.

D-Peptide and D-Protein Technology: Recent Advances, Challenges, and Opportunities.

Total chemical protein synthesis provides access to entire D-protein enantiomers enabling unique applications in molecular biology, structural biology, and bioactive compound discovery. Key enzymes involved in the central dogma of molecular biology have been prepared in their D-enantiomeric forms facilitating the development of mirror-image life. Crystallization of a racemic mixture of L- and D-protein enantiomers provides access to high-resolution X-ray structures of polypeptides. Additionally, D-enantiomers of protein drug targets can be used in mirror-image phage display allowing discovery of non-proteolytic D-peptide ligands as lead candidates. This review discusses the unique applications of D-proteins including the synthetic challenges and opportunities.

Towards the use of an amino acid cleavable linker for solid-phase chemical synthesis of peptides and proteins.

The synthesis of proteins by solid-phase chemical ligation (SPCL) suffers from the paucity of linkers that can be cleaved under mild conditions. Here, we deployed a spontaneous nickel-assisted cleavage (SNAC) tag, known to undergo spontaneous cleavage in the presence of nickel(II), as a linker for C-to-N SPCL.

Spatio-Temporal Photoactivation of Cytotoxic Proteins.

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.

Cryo-kinetics Reveal Dynamic Effects on the Chemistry of Human Dihydrofolate Reductase.

Effects of isotopic substitution on the rate constants of human dihydrofolate reductase (HsDHFR), an important target for anti-cancer drugs, have not previously been characterized due to its complex fast kinetics. Here, we report the results of cryo-measurements of the kinetics of the HsDHFR catalyzed reaction and the effects of protein motion on catalysis. Isotopic enzyme labeling revealed an enzyme KIE (kHLE /kHHE ) close to unity above 0 °C; however, the enzyme KIE was increased to 1.72±0.15 at -20 °C, indicating that the coupling of protein motions to the chemical step is minimized under optimal conditions but enhanced at non-physiological temperatures. The presented cryogenic approach provides an opportunity to probe the kinetics of mammalian DHFRs, thereby laying the foundation for characterizing their transition state structure.

Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency.

Genetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

Asparaginyl endopeptidases: enzymology, applications and limitations.

Asparaginyl endopeptidases (AEP) are cysteine proteases found in mammalian and plant cells. Several AEP isoforms from plant species were found to exhibit transpeptidase activity which is integral for the key head-to-tail cyclisation reaction during the biosynthesis of cyclotides. Since many plant AEPs exhibit excellent enzyme kinetics for peptide ligation via a relatively short substrate recognition sequence, they have become appealing tools for peptide and protein modification. In this review, research focused on the enzymology of AEPs and their applications in polypeptide cyclisation and labelling will be presented. Importantly, the limitations of using AEPs and opportunities for future research and innovation will also be discussed.

Approaches for peptide and protein cyclisation.

The cyclisation of polypeptides can play a crucial role in exerting biological functions, maintaining stability under harsh conditions and conferring proteolytic resistance, as demonstrated both in nature and in the laboratory. To date, various approaches have been reported for polypeptide cyclisation. These approaches range from the direct linkage of N- and C- termini to the connection of amino acid side chains, which can be applied both in reaction vessels and in living systems. In this review, we categorise the cyclisation approaches into chemical methods (e.g. direct backbone cyclisation, native chemical ligation, aldehyde-based ligations, bioorthogonal reactions, disulphide formation), enzymatic methods (e.g. subtiligase variants, sortases, asparaginyl endopeptidases, transglutaminases, non-ribosomal peptide synthetases) and protein tags (e.g. inteins, engineered protein domains for isopeptide bond formation). The features of each approach and the considerations for selecting an appropriate method of cyclisation are discussed.

The role of streptavidin and its variants in catalysis by biotinylated secondary amines.

Here, we combine the use of host screening, protein crystallography and QM/MM molecular dynamics simulations to investigate how the protein structure affects iminium catalysis by biotinylated secondary amines in a model 1,4 conjugate addition reaction. Monomeric streptavidin (M-Sav) lacks a quaternary structure and the solvent-exposed reaction site resulted in poor product conversion in the model reaction with low enantio- and regioselectivities. These parameters were much improved when the tetrameric host T-Sav was used; indeed, residues at the symmetrical subunit interface were proven to be critical for catalysis through a mutagenesis study. The use of QM/MM simulations and the asymmetric dimeric variant D-Sav revealed that both Lys121 residues which are located in the hosting and neighboring subunits play a critical role in controlling the stereoselectivity and reactivity. Lastly, the D-Sav template, though providing a lower conversion than that of the symmetric tetrameric counterpart, is likely a better starting point for future protein engineering because each surrounding residue within the asymmetric scaffold can be refined for secondary amine catalysis.

Condensation of 2-((Alkylthio)(aryl)methylene)malononitrile with 1,2-Aminothiol as a Novel Bioorthogonal Reaction for Site-Specific Protein Modification and Peptide Cyclization.

Site-specific modification of peptides and proteins has wide applications in probing and perturbing biological systems. Herein we report that 1,2-aminothiol can react rapidly, specifically and efficiently with 2-((alkylthio)(aryl)methylene)malononitrile (TAMM) under biocompatible conditions. This reaction undergoes a unique mechanism involving thiol-vinyl sulfide exchange, cyclization, and elimination of dicyanomethanide to form 2-aryl-4,5-dihydrothiazole (ADT) as a stable product. An 1,2-aminothiol functionality can be introduced into a peptide or a protein as an N-terminal cysteine or an unnatural amino acid. The bioorthogonality of this reaction was demonstrated by site-specific labeling of not only synthetic peptides and a purified recombinant protein but also proteins on mammalian cells and phages. Unlike other reagents in bioorthogonal reactions, the chemical and physical properties of TAMM can be easily tuned. TAMM can also be applied to generate phage-based ADT-cyclic peptide libraries without reducing phage infectivity. Using this approach, we identified ADT-cyclic peptides with high affinity to different protein targets, providing valuable tools for biological studies and potential therapeutics. Furthermore, the mild reaction conditions of TAMM condensation warrant its use with other bioorthogonal reactions to simultaneously achieve multiple site-specific modifications.

Exploring the Chemoselectivity towards Cysteine Arylation by Cyclometallated AuIII Compounds: New Mechanistic Insights.

To gain more insight into the factors controlling efficient cysteine arylation by cyclometallated AuIII complexes, the reaction between selected gold compounds and different peptides was investigated by high-resolution liquid chromatography electrospray ionization mass spectrometry (HR-LC-ESI-MS). The deduced mechanisms of C-S cross-coupling, also supported by density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) calculations, evidenced the key role of secondary peptidic gold binding sites in favouring the process of reductive elimination.

Applying switchable Cas9 variants to in vivo gene editing for therapeutic applications.

Progress in targeted gene editing by programmable endonucleases has paved the way for their use in gene therapy. Particularly, Cas9 is an endonuclease with high activity and flexibility, rendering it an attractive option for therapeutic applications in clinical settings. Many disease-causing mutations could potentially be corrected by this versatile new technology. In addition, recently developed switchable Cas9 variants, whose activity can be controlled by an external stimulus, provide an extra level of spatiotemporal control on gene editing and are particularly desirable for certain applications. Here, we discuss the considerations and difficulties for implementing Cas9 to in vivo gene therapy. We put particular emphasis on how switchable Cas9 variants may resolve some of these barriers and advance gene therapy in the clinical setting.

Streptavidin-Hosted Organocatalytic Aldol Addition.

In this report, the streptavidin-biotin technology was applied to enable organocatalytic aldol addition. By attaching pyrrolidine to the valeric motif of biotin and introducing it to streptavidin (Sav), a protein-based organocatalytic system was created, and the aldol addition of acetone with p-nitrobenzaldehyde was tested. The conversion of substrate to product can be as high as 93%. Although the observed enantioselectivity was only moderate (33:67 er), further protein engineering efforts can be included to improve the selectivity. These results have proven the concept that Sav can be used to host stereoselective aldol addition.

Heavy Enzymes and the Rational Redesign of Protein Catalysts.

An unsolved mystery in biology concerns the link between enzyme catalysis and protein motions. Comparison between isotopically labelled "heavy" dihydrofolate reductases and their natural-abundance counterparts has suggested that the coupling of protein motions to the chemistry of the catalysed reaction is minimised in the case of hydride transfer. In alcohol dehydrogenases, unnatural, bulky substrates that induce additional electrostatic rearrangements of the active site enhance coupled motions. This finding could provide a new route to engineering enzymes with altered substrate specificity, because amino acid residues responsible for dynamic coupling with a given substrate present as hotspots for mutagenesis. Detailed understanding of the biophysics of enzyme catalysis based on insights gained from analysis of "heavy" enzymes might eventually allow routine engineering of enzymes to catalyse reactions of choice.

Crystal Structure and Biophysical Analysis of Furfural-Detoxifying Aldehyde Reductase from Clostridium beijerinckii.

Many aldehydes, such as furfural, are present in high quantities in lignocellulose lysates and are fermentation inhibitors, which makes biofuel production from this abundant carbon source extremely challenging. Cbei_3974 has recently been identified as an aldo-keto reductase responsible for partial furfural resistance in Clostridium beijerinckii Rational engineering of this enzyme could enhance the furfural tolerance of this organism, thereby improving biofuel yields. We report an extensive characterization of Cbei_3974 and a single-crystal X-ray structure of Cbei_3974 in complex with NADPH at a resolution of 1.75 Å. Docking studies identified residues involved in substrate binding, and an activity screen revealed the substrate tolerance of the enzyme. Hydride transfer, which is partially rate limiting under physiological conditions, occurs from the pro-R hydrogen of NADPH. Enzyme isotope labeling revealed a temperature-independent enzyme isotope effect of unity, indicating that the enzyme does not use dynamic coupling for catalysis and suggesting that the active site of the enzyme is optimally configured for catalysis with the substrate tested.IMPORTANCE Here we report the crystal structure and biophysical properties of an aldehyde reductase that can detoxify furfural, a common inhibitor of biofuel fermentation found in lignocellulose lysates. The data contained here will serve as a guide for protein engineers to develop improved enzyme variants that would impart furfural resistance to the microorganisms used in biofuel production and thus lead to enhanced biofuel yields from this sustainable resource.

ß1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel.

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory ß-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or ß1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) ß1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the ß1-subunit within the α/ß1-subunit complex.

Reaction Mechanism of Organocatalytic Michael Addition of Nitromethane to Cinnamaldehyde: A Case Study on Catalyst Regeneration and Solvent Effects.

The Michael addition of nitromethane to cinnamaldehyde has been computationally studied in the absence of a catalyst and the presence of a biotinylated secondary amine by a combined computational and experimental approach. The calculations were performed at the density functional theory (DFT) level with the M06-2X hybrid functional, and a polarizable continuum model has been employed to mimic the effect of two different solvents: dichloromethane (DCM) and water. Contrary to common assumption, the product-derived iminium intermediate was absent in both of the solvents tested. Instead, hydrating the C1-C2 double bond in the enamine intermediate directly yields the tetrahedral intermediate, which is key for forming the product and regenerating the catalyst. Enamine hydration is concerted and found to be rate-limiting in DCM but segregated into two non-rate-limiting steps when the solvent is replaced with water. However, further analysis revealed that the use of water as solvent also raises the energy barriers for other chemical steps, particularly the critical step of C-C bond formation between the iminium intermediate and nucleophile; this consequently lowers both the reaction yield and enantioselectivity of this LUMO-lowering reaction, as experimentally detected. These findings provide a logical explanation to why water often enhances organocatalysis when used as an additive but hampers the reaction progress when employed as a solvent.

Reactivity and Selectivity of Iminium Organocatalysis Improved by a Protein Host.

There has been growing interest in performing organocatalysis within a supramolecular system as a means of controlling reaction reactivity and stereoselectivity. Here, a protein is used as a host for iminium catalysis. A pyrrolidine moiety is covalently linked to biotin and introduced to the protein host streptavidin for organocatalytic activity. Whereas in traditional systems stereoselectivity is largely controlled by the substituents added to the organocatalyst, enantiomeric enrichment by the reported supramolecular system is completely controlled by the host. Also, the yield of the model reaction increases over 10-fold when streptavidin is included. A 1.1 Šcrystal structure of the protein-catalyst complex and molecular simulations of a key intermediate reveal the chiral scaffold surrounding the organocatalytic reaction site. This work illustrates that proteins can be an excellent supramolecular host for driving stereoselective secondary amine organocatalysis.

Reduction of Folate by Dihydrofolate Reductase from Thermotoga maritima.

Mammalian dihydrofolate reductases (DHFRs) catalyze the reduction of folate more efficiently than the equivalent bacterial enzymes do, despite typically having similar efficiencies for the reduction of their natural substrate, dihydrofolate. In contrast, we show here that DHFR from the hyperthermophilic bacterium Thermotoga maritima can catalyze reduction of folate to tetrahydrofolate with an efficiency similar to that of reduction of dihydrofolate under saturating conditions. Nuclear magnetic resonance and mass spectrometry experiments showed no evidence of the production of free dihydrofolate during either the EcDHFR- or TmDHFR-catalyzed reductions of folate, suggesting that both enzymes perform the two reduction steps without release of the partially reduced substrate. Our results imply that the reaction proceeds more efficiently in TmDHFR than in EcDHFR because the more open active site of TmDHFR facilitates protonation of folate. Because T. maritima lives under extreme conditions where tetrahydrofolate is particularly prone to oxidation, this ability to salvage folate may impart an advantage to the bacterium by minimizing the squandering of a valuable cofactor.