Ibrahim Yildiz

Peptides have become an indispensable tool in engineering of multifunctional nanostructure platforms for biomedical applications such as targeted drug and gene delivery, imaging and biosensing. They can be covalently incorporated into a variety of nanoparticles (NPs) including polymers, metallic nanoparticles, and others. Using different bioconjugation techniques, multifunctional peptide‐modified NPs can be formulated to produce therapeutical and diagnostic platforms offering high specificity, lower toxicity, biocompatibility, and stimuli responsive behavior. Targeting peptides can direct the nanoparticles into specific tissues for targeted drug and gene delivery and imaging applications due to their specificity towards certain receptors. Furthermore, due to their stimuli‐responsive features, they can offer controlled release of therapeutics into desired sites of disease. In addition, peptide‐based biosensors and imaging agents can provide non‐invasive detection and monitoring of diseases including cancer, infectious diseases, and neurological disorders. In this review, we covered the design and formulation of recent peptide‐based NP platforms, as well as their utilization in in vitro and in vivo applications such as targeted drug and gene delivery, targeting, sensing, and imaging applications. In the end, we provided the future outlook to design new peptide conjugated nanomaterials for biomedical applications.

L-Proline dehydrogenase (ProDH) is a flavin-dependent oxidoreductase, which catalyzes the oxidation of L-proline to (S)-1-pyrroline-5-carboxylate. Based on the experimental studies, a stepwise proton and hydride transfer mechanism is supported. According to this mechanism, the amino group of L-proline is deprotonated by a nearby Lys residue, which is followed by the hydride transfer process from C5 position of L-proline to N5 position of isoalloxazine ring of FAD. It was concluded that the hydride transfer step is rate limiting in the reductive half-reaction, however, in the overall reaction, the oxidation of FAD is the rate limiting step. In this study, we performed a computational mechanistic investigation based on ONIOM method to elucidate the mechanism of the reductive half-reaction corresponding to the oxidation of L-proline into iminoproline. Our calculations support the stepwise mechanism in which the deprotonation occurs initially as a fast step as result of a proton transfer from L-proline to the Lys residue. Subsequently, a hydride ion transfers from L-proline to FAD with a higher activation barrier. The enzyme-product complex showed a strong interaction between reduced FAD and iminoproline, which might help to explain why a step in the oxidative half-reaction is rate-limiting.

D‐Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) is an amine oxidase which catalyzes the conversion of D‐arginine into iminoarginine. It contains a non‐covalent FAD cofactor that is involved in the oxidation mechanism. Based on substrate, solvent, and multiple kinetic isotope effects studies, a stepwise hydride transfer mechanism is proposed. It was shown that D‐arginine binds to the active site of enzyme as α‐amino group protonated, and it is deprotonated before a hydride ion is transferred from its α‐C to FAD. Based on a mutagenesis study, it was concluded that a water molecule is the most likely catalytic base responsible from the deprotonation of α‐amino group. In this study, we formulated computational models based on ONIOM method to elucidate the oxidation mechanism of D‐arginine into iminoarginine using the crystal structure of enzyme complexed with iminoarginine. The calculations showed that Arg222, Arg305, Tyr249, Glu87, His 48, and two active site water molecules play key roles in binding and catalysis. Model systems showed that the deprotonation step occurs prior to hydride transfer step, and active site water molecule(s) may have participated in the deprotonation process.

L-Aspartate oxidase (Laspo) is responsible for the oxidation of l-aspartate into iminoaspartate using flavin as a cofactor. During this process flavin is reduced, and it can be reoxidized by either molecular oxygen or fumarate. The overall fold and the catalytic residues of Laspo are similar to succinate dehydrogenase and fumarate reductase. On the basis of deuterium kinetic isotope effects as well as other kinetic and structural data, it is proposed that the enzyme can catalyze the oxidation of l-aspartate through a mechanism similar to amino acid oxidases. It is suggested that a proton is removed from the α-amino group, while a hydride is transferred from C2 to flavin. It is also suggested that the hydride transfer is a rate-limiting step. However, there is still an ambiguity about the stepwise or concerted mechanism of hydride- and proton-transfer steps. In this study, we formulated some computational models to study the hydride-transfer mechanism using the crystal structure of Escherichia colil-aspartate oxidase in complexes with succinate. The calculations involved our own N-layered integrated molecular orbital and molecular mechanics method, and we evaluated the geometry and energetics of the hydride/proton-transfer processes while probing the roles of active site residues. Based on the calculations, it is concluded that proton- and hydride-transfer steps are decoupled, and a stepwise mechanism might be operative as opposed to the concerted one.

Notum is a member of serine hydrolyses that cleaves the palmitoleate moiety from Wingless-related integration site (Wnt) ligands. This enzyme plays crucial functions through modulating the Wnt signaling pathway. Inhibition of Notum carries therapeutic effects against a number of maladies including osteoporosis, cancer, and Alzheimer’s disease. Recently, a class of irreversible inhibitors based on esters of 4-(indolin-1-yl)-4-oxobutanoic acid have been reported. Using the crystal structures of enzyme-4-(indolin-1-yl)-4-oxobutanoate adduct and 4-(indolin-1-yl)-4-oxobutanoic acid-enzyme complex, we studied computationally the proposed inhibition mechanism using model systems based on the own n-layered integrated molecular orbital and molecular mechanics (ONIOM) method. In the first place, model systems were formulated to investigate the transesterification between the catalytic serine residue, Ser-232, and the methyl ester of 4-(indolin-1-yl)-4-oxobutanoate. In the second place, the hydrolysis mechanism of the resultant enzyme–inhibitor adduct was studied. The energetics of these steps were analyzed using a density functional theory functional in the ONIOM method. In addition, the roles of active-site residues during these steps were highlighted. It was found that the hydrolysis of the covalent adduct is highly endergonic corroborating the irreversible inhibition mechanism. These results will shed light not only on the inhibition mechanism but also on the catalytic mechanism.

Histone deacetylase 10 (HDAC 10) catalyzes deacetylation of N⁸-acetylspermidine into spermidine in the cytosolic region of eukaryotic cells. Inhibition of HDAC 10 has clinical importance in certain types of cancers. Recently, X-ray crystal structures corresponding to the substrate-bound, tetrahedral intermediate-bound, and product-bound enzymes have been resolved using variant forms of humanized HDAC 10. Based on these structures, it was proposed that Y307 residue polarizes the carbonyl of the acetyl group in N⁸-acetylspermidine together with a zinc atom, which is coordinated by D174, H176, D267, and an H₂O molecule. The H₂O molecule undergoes nucleophilic addition to the carbonyl carbon of N⁸-acetylspermidine to form the tetrahedral intermediate. During this process, it is suggested that H136 acts as a general base to deprotonate the H₂O molecule. It is further proposed that the protonation of the amide N atom of the tetrahedral intermediate by H137 causes the deacetylation forming the final products, spermidine and acetate ion. In this study, computational models based on the ONIOM method were employed to study the proposed mechanism for the two steps of the deacetylation process based on the crystal structure of the substrate-bound enzyme. The energy profiles of each step as well as the roles of the active site residues were investigated for the catalysis. The calculated activation barrier is in good agreement with the reported k_cat value.

Molecular catalysts are acknowledged for the ability to design reaction sites within well‐defined structures to achieve high catalytic activities. However, in many cases, molecular catalysts undergo structural changes to some other form(s), which are finally the real catalysts. Here, we report two half‐sandwich ruthenium complexes; [Ru([9]aneN3)(bpy)Cl]⁺ 3 and [Ru([9]aneN3)(pic)OH₂]²⁺ 4, based on the macrocyclic 1,4,7‐triazacyclononane ([9]aneN3) ligand for water oxidation (WO). The two complexes have similar core but different ancillary ligands, which greatly affected their stability as well as activity for WO. Complex 3 retained high stability and excellent activity (turnover number [TON] = 1250) in chemical WO and first‐order reaction kinetics with respect to [Ce^IV] with a calculated rate constant (k_cat) of 34.59 s⁻¹. Further, the complex demonstrated very low overpotential of ~210 mV in electrochemical WO. At an overpotential of only 400 mV, turnover frequency (TOF) of complex 3 was electrochemically estimated to be 131.2 s⁻¹. In contrast, complex 4 underwent picolinate ligand dissociation, as a deactivation pathway, to form the tri‐aqua derivative. Density functional theory (DFT) calculations are used to explain the dissociation mechanism of picolinate ligand in complex 4, which happens through a stepwise dissociation mechanism.