Emergence of a short peptide based reductase via activation of the model hydride rich cofactor

Extant biological systems utilize complex enzymes and low molecular weight cofactors (like dihydronicotinamides) for efficient hydride transfer and electron carrier functions in crucial metabolic processes. However, these cofactors are generally inefficient without the structural support of enzymes. Early metabolic pathways likely employed simpler energy harvesting methods, exploiting energetically favorable electron transfer from low molecular weight cofactors to generate proton gradients necessary for pyrophosphate synthesis. Primitive catalysts would have assisted these early oxidoreduction reactions, enabling minimal metabolic cycles. Contemporary biology relies on cofactors such as NADPH and NADH, but these require the precisely structured binding pockets of evolved enzymes for effective hydride transfer. A key gap remains in understanding how early collections of cofactors and biopolymers facilitated these processes. Short peptides, forming amyloid phases with solvent-exposed amino acid residues, are potential surrogates for primitive protein folds. These oligomeric peptides can create robust nanostructures even under harsh conditions. This study investigates whether short peptides could leverage electrostatic interactions and binding sites with charged residues to perform hydride transfer with rudimentary cofactors, facilitating reactions otherwise unfavorable in aqueous environments.

The paper draws upon existing research on the role of cofactors in modern metabolism (e.g., NADPH, NADH in glycolysis and oxidative phosphorylation), the importance of enzyme active sites in cofactor activation, and the potential of short peptides and amyloid structures as models for early life catalytic systems. It cites studies suggesting that amyloid structures may have been critical components of early life, and that their capacity for self-assembly and molecular interactions make them suitable candidates for catalytic activity in prebiotic environments. Previous work on peptide-based assemblies for catalytic activity and the use of NaBH₄ as a model hydride transfer agent are also referenced. The limitations of using NaBH₄ as a model for biological hydride transfer agents are acknowledged and discussed.

The research employed a short peptide fragment (LVFFA) from the Aβ amyloid (1-42) nucleating core as a starting point, modifying it by adding arginine and leucine residues to create Ac-RLVFFAL-NH₂ (ARG-16). The self-assembly of this peptide into nanotubes was characterized using techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), and Thioflavin T (ThT) fluorescence. The binding efficiency of the nanotubes for small molecules was assessed using a fluorescent dye (FITC) and negatively charged gold nanoparticles (GNPneg). The catalytic reduction of a model hydrophobic substrate, hexyl 4-nitrobenzoate (1), to 4-nitrobenzyl alcohol (2) in the presence of NaBH₄ was investigated. The effects of pH, peptide morphology (using HFIP to disassemble nanotubes and Na₂SO₄ to induce bundling), and sequence variations (modifying the hydrophobic core and substituting arginine with other residues like lysine, histidine, and glutamic acid) were explored. To enhance catalytic activity, a quaternized glycine (betaine) was introduced at the N-terminus (BET-16), and the resulting nanotubes were characterized and tested for catalytic activity. The reusability and substrate selectivity of the peptide assemblies were also examined, along with the potential for controlled reduction of a bis-ester. Techniques such as HPLC, zeta potential measurements, ¹¹B NMR, and SEM-EDS were used for detailed characterization and analysis.

The ARG-16 peptide self-assembled into nanotubes with exposed cationic and hydrophobic surfaces. These nanotubes facilitated the reduction of hexyl 4-nitrobenzoate (1) in the presence of NaBH₄, a process not observed without the peptide assemblies or NaBH₄ alone. The reaction yield was significantly enhanced with BET-16, a modified peptide with quaternized glycine, reaching approximately 60.5 ± 11% compared to 27.5 ± 4% for ARG-16. This improvement is attributed to the stronger electrostatic interactions between the supercharged BET-16 surface and the BH₄⁻ ion. ¹¹B NMR confirmed the presence and stability of NaBH₄ bound to the BET-16 nanotubes. The system displayed substrate selectivity, reducing different nitrobenzoates with varying efficiencies depending on the length of their alkyl chains and polarity. The BET-16 system performed controlled reduction of a bis-ester, producing a monoester as an intermediate product, unlike LiAlH₄, which reduced both ester groups. The peptide assemblies exhibited reusability, maintaining catalytic activity over three consecutive cycles. Control experiments with scrambled peptides, peptides with different amino acid substitutions, and disassembled or bundled nanotubes demonstrated the importance of the specific peptide sequence, morphology (nanotubes), and exposed cationic surfaces for catalytic activity.

The study's findings demonstrate that short peptide assemblies can effectively mimic some aspects of enzyme function, binding and activating a weak hydride transfer agent (NaBH₄) to catalyze a challenging reduction reaction in water. The use of NaBH₄ as a model cofactor is a simplification, and the results may not be directly transferable to biological systems with more complex cofactors. The increased efficiency of BET-16 highlights the importance of highly cationic surfaces for cofactor binding and activation. The observed substrate selectivity and controlled reduction capability suggest that these peptide assemblies can achieve precise reaction control. The demonstrated reusability is a significant advantage from both fundamental and application perspectives. The results support the hypothesis that short peptides could have played a significant role in early metabolic processes, showcasing a potential pathway for the emergence of more complex biocatalysts. The study provides insights into the design of novel functional nanomaterials with adaptable catalytic properties.

This research successfully demonstrates the creation of short peptide-based amyloid nanotubes capable of binding and activating a model hydride transfer agent to catalyze the reduction of ester substrates in water. The enhanced efficiency achieved with a supercharged peptide variant and the observed substrate selectivity, controlled reduction, and reusability highlight the potential of these minimal systems as catalysts. The findings contribute significantly to our understanding of the emergence of protometabolism and suggest new directions for the development of advanced functional nanomaterials with potential applications in various fields. Future research could focus on exploring the interactions with more biologically relevant cofactors and substrates, as well as investigating the potential for designing more sophisticated peptide-based catalysts with broader catalytic capabilities.

The study uses a model hydride transfer agent (NaBH₄), which differs significantly from the complex biological cofactors used in contemporary metabolism. The reaction conditions (pH 10) are not physiological, which limits the immediate biological relevance of the findings. While the system shows reusability, the long-term stability and the effects of repeated use on the structure and catalytic activity require further investigation. The range of substrates tested is limited, and future studies should explore a broader substrate scope to better understand the substrate specificity and selectivity of the peptide assemblies. Finally, the transition from model reactions to biologically relevant processes and environments needs additional research.