Comparison of the Backbone Dynamics of Dehaloperoxidase-Hemoglobin Isoenzymes.

Dehaloperoxidase (DHP) is a multifunctional hemeprotein with a functional switch generally regulated by the chemical class of the substrate. Its two isoforms, DHP-A and DHP-B, differ by only five amino acids and have an almost identical protein fold. However, the catalytic efficiency of DHP-B for oxidation by a peroxidase mechanism ranges from 2- to 6-fold greater than that of DHP-A depending on the conditions. X-ray crystallography has shown that many substrates and ligands have nearly identical binding in the two isoenzymes, suggesting that the difference in catalytic efficiency could be due to differences in the conformational dynamics. We compared the backbone dynamics of the DHP isoenzymes at pH 7 through heteronuclear relaxation dynamics at 11.75, 16.45, and 19.97 T in combination with four 300 ns MD simulations. While the overall dynamics of the isoenzymes are similar, there are specific local differences in functional regions of each protein. In DHP-A, Phe35 undergoes a slow chemical exchange between two conformational states likely coupled to a swinging motion of Tyr34. Moreover, Asn37 undergoes fast chemical exchange in DHP-A. Given that Phe35 and Asn37 are adjacent to Tyr34 and Tyr38, it is possible that their dynamics modulate the formation and migration of the active tyrosyl radicals in DHP-A at pH 7. Another significant difference is that both distal and proximal histidines have a 15-18% smaller S2 value in DHP-B, thus their greater flexibility could account for the higher catalytic activity. The distal histidine grants substrate access to the distal pocket. The greater flexibility of the proximal histidine could also accelerate H2O2 activation at the heme Fe by increased coupling of an amino acid charge relay to stabilize the ferryl Fe(IV) oxidation state in a Poulos-Kraut "push-pull"-type peroxidase mechanism.

Histidine tag modified magnetic beads for analysis of arsenic binding proteins.

BACKGROUND: Many proteins with thiol groups can bind with trivalent arsenic which are termed as arsenic binding proteins, thus change their physiological functions. Therefore, it is vital to analyze the arsenic binding proteins in cells. The Pull-Down strategy based on biotinylated phenylarsenic acid (Bio-PAO(III)) probes is an effective way for analysis of arsenic binding proteins. In this strategy, streptavidin magnetic beads (SA-MBs) was applied to capture the arsenic binding proteins conjugating with Bio-PAO(III) probe. However, strong interaction between SA and biotin makes the elution of arsenic binding proteins not easy. RESULTS: We developed a novel affinity separation strategy to address the challenge of eluting arsenic binding proteins, a key issue with the existing Bio-PAO(III) Pull-Down method. By employing magnetic beads modified with Nα-Bis(carboxymethyl)-l-lysine (NTA-Lys), polyhistidine-tag (His6-Tag), and SA (MB-NTA(Ni)-His6-SA), we established a more efficient purification process. This innovative approach enables selective capture of arsenic binding proteins in HepG2 cells labeled by Bio-PAO(III) probes, facilitating gentle digestion by trypsin for precise identification through capillary high performance liquid chromatography (Cap HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). What is more, the magnetic beads can be regenerated by using imidazole as the eluent, and the obtained MB-NTA(Ni) can be reloaded with His6-SA for next use. Our method successfully identified 41 arsenic binding proteins, including those involved in cytoskeletal structure, heat shock response, transcriptional regulation, DNA damage repair, redox state regulation, mitochondrial dehydrogenase function, and protein synthesis and structure. SIGNIFICANCE: This work contributes to a more comprehensive understanding of the toxic mechanisms of arsenic, potentially providing valuable insights for the prevention or treatment of arsenic-related diseases.

A novel EGCG-Histidine complex improves gelling and physicochemical properties of porcine myofibrillar proteins: Insight into underlying mechanisms.

Herein, an EGCG-Histidine complex is prepared, characterized, and further used to improve gel properties of myofibrillar proteins (MP). Results of FTIR, XRD, UV-Vis spectroscopy showed that histidine is covalently bound to EGCG by Michael addition or Schiff base reaction to form EGCG-Histidine complex, and antioxidant activity of EGCG-Histidine complex is significantly increased compared to EGCG or histidine alone (P < 0.05). The addition of EGCG-Histidine complex results in cooking loss of gel decreasing from 66.7 ± 0.23 % to 40.3 ± 2.02 %, and improves rheological properties of MP, and enhances gel strength from 0.10 ± 0.01 N to 0.22 ± 0.03 N, indicating positive effect of EGCG-Histidine complex on MP gel formation, above results is supported by results of SEM, CD spectroscopy, SDS-PAGE, and tryptophan fluorescence. These results indicated that EGCG-Histidine complex can be used as a functional ingredient to improve gel quality of meat products.

Co-Assembly of Cancer Drugs with Cyclo-HH Peptides: Insights from Simulations and Experiments.

Peptide-based nanomaterials can serve as promising drug delivery agents, facilitating the release of active pharmaceutical ingredients while reducing the risk of adverse reactions. We previously demonstrated that Cyclo-Histidine-Histidine (Cyclo-HH), co-assembled with cancer drug Epirubicin, zinc, and nitrate ions, can constitute an attractive drug delivery system, combining drug self-encapsulation, enhanced fluorescence, and the ability to transport the drug into cells. Here, we investigated both computationally and experimentally whether Cyclo-HH could co-assemble, in the presence of zinc and nitrate ions, with other cancer drugs with different physicochemical properties. Our studies indicated that Methotrexate, in addition to Epirubicin and its epimer Doxorubicin, and to a lesser extent Mitomycin-C and 5-Fluorouracil, have the capacity to co-assemble with Cyclo-HH, zinc, and nitrate ions, while a significantly lower propensity was observed for Cisplatin. Epirubicin, Doxorubicin, and Methorexate showed improved drug encapsulation and drug release properties, compared to Mitomycin-C and 5-Fluorouracil. We demonstrated the biocompatibility of the co-assembled systems, as well as their ability to intracellularly release the drugs, particularly for Epirubicin, Doxorubicin, and Methorexate. Zinc and nitrate were shown to be important in the co-assembly, coordinating with drugs and/or Cyclo-HH, thereby enabling drug-peptide as well as drug-drug interactions in successfully formed nanocarriers. The insights could be used in the future design of advanced cancer therapeutic systems with improved properties.

Histidine Protonation States Regulate the State Transition from R State Hemoglobin.

The objective of our work is to investigate the impact of pH on the structural changes of hemoglobin that affect its O2 affinity, known as the Bohr effect. We conducted molecular dynamics (MD) simulations to explore the transition between various hemoglobin states based on the protonation states (PSs) of two histidine residues (ßHis143 and ßHis146). We conducted the MD simulations from the R and R2 states with three sets of PSs assuming pH values of 7.0, 6.5, and 5.5, aiming to investigate the influence of pH on hemoglobin behavior. Our results demonstrated that the protonated His residues promote the state transition from the R state to the R2 state and encourage elongation of the distance between the ß1-ß2 subunits by weakening the inter-subunit interactions in the R state. These observations, aligning with the experimental evidence, indicate that the R2 state typically crystallizes under low pH conditions. Our findings suggest that the relationship between the PSs and the structural stability of the R state plays a role in the acid and alkaline Bohr effect.

Beyond copper: examining the significance of His-mutations in mycobacterial GroEL1 HRCT for Ni(II) complex stability and formation.

Recently, we have studied the coordination chemistry of the Cu(II)-histidine-rich C-terminal tail (HRCT) complex of the mycobacterial GroEL1 protein. The structure of this domain differs significantly compared to the well-known methionine-glycine-rich GroEL chaperonin - it was predicted that mycobacterial GroEL1 could play a significant role in the metal homeostasis of Mycobacteria, especially copper. However, we found that this particular domain's pattern also repeats in a number of Ni(II)-binding proteins. Here, we present the studies concerning the properties of GroEL1 HRCT as a ligand for Ni(II) ions. For this purpose, we chose eight model peptides: L1 - Ac-DHDHHHGHAH, L2 - Ac-DKPAKAEDHDHHHGHAH, and 6 mutants of the latter in the pH range of 2-11. We examined the stoichiometry, stability, and spectroscopic features of copper complexes. We noticed that similar to the Cu(II)-complex, the presence of a Lys5 residue significantly increases the stability of the system. The impact of His mutations was also examined and carefully studied using NMR spectroscopy. His9 and His13 are the crucial residues for Ni(II) binding, whereas His12 has minimal relevance in complex formation.

The green cupredoxin CopI is a multicopper protein able to oxidize Cu(I).

Anthropogenic activities in agriculture and health use the antimicrobial properties of copper. This has led to copper accumulation in the environment and contributed to the emergence of copper resistant microorganisms. Understanding bacterial copper homeostasis diversity is therefore highly relevant since it could provide valuable targets for novel antimicrobial treatments. The periplasmic CopI protein is a monodomain cupredoxin comprising several copper binding sites and is directly involved in copper resistance in bacteria. However, its structure and mechanism of action are yet to be determined. To study the different binding sites for cupric and cuprous ions and to understand their possible interactions, we have used mutants of the putative copper binding modules of CopI and spectroscopic methods to characterize their properties. We show that CopI is able to bind a cuprous ion in its central histidine/methionine-rich region and oxidize it thanks to its cupredoxin center. The resulting cupric ion can bind to a third site at the N-terminus of the protein. Nuclear magnetic resonance spectroscopy revealed that the central histidine/methionine-rich region exhibits a dynamic behavior and interacts with the cupredoxin binding region. CopI is therefore likely to participate in copper resistance by detoxifying the cuprous ions from the periplasm.

Exploring binding preferences: Cu(II), Ni(II), and Zn(II) complexes of mycobacterial GroEL1 His-rich and Glu/His-rich domains.

Mycobacterial histidine-rich GroEL1 protein significantly differs from the well-known methionine-glycine-rich GroEL chaperonin and most preferably participates in Cu(II) homeostasis. Some GroEL1 proteins, however, do not possess six but only three histidine residues and more acidic residues that can function as binding sites for metal ions. To evaluate the importance of this difference, we examined and compared the properties of GroEL1 His-rich or Glu/His-rich C-terminal domains as ligands for Cu(II), Ni(II), and Zn(II) ions. We studied the stoichiometry, stability, and binding sites of Cu(II)/Ni(II)/Zn(II) complexes of two model peptides: XEN = Ac-DKPEEEEDGHGHAH (M. xenopi) and ABS = Ac-DKPAEEADHGHGHHGHAH (M. abscessus) in the pH range 2-11. In the case of Cu(II), Ni(II), and Zn(II) complexes of XEN and ABS, ABS always formed more stable complexes. For XEN, there seemed to be no preference for Ni(II) or Zn(II) ions. In contrast, for ABS, Zn(II) formed a complex that was slightly more stable than the one formed by Ni(II). This may be due to the 6 His residues, which preferentially interact with Zn(II) rather than Ni(II). The study identified that an equilibrium of complexes-known as polymorphism-may occur in ABS complexes. Therefore, distinct sets of histidine residues may be involved in metal binding.

Novel melanin-derived stationary phase for immobilized metal ion affinity chromatography in recombinant His-tagged protein purification.

The matrix of the stationary phase is a crucial element in affinity chromatography for protein purification. Various materials, including polymer or magnetic materials, have been employed as the matrix in the purification of His-tagged protein. Here, for the first time, we utilized a combination of melanin and alginate, both natural polymer materials, to synthesize Ni-melanin/alginate (Ni-M/A) beads for His-tagged protein purification. We investigated the binding of His-tagged Mpro on the Ni-M/A beads, referred to as Ni-M/A-Mpro, and assessed the elution efficiency of Mpro from the beads. Our examination involved FTIR, EDS, XRD, SDS-PAGE, and Western blotting methods. FTIR spectra revealed notable changes in the stretching patterns and intensities of hydroxyl, amine, carbonyl, imine and amide chemical groups, when Mpro protein was present in the Ni-M/A sample. XRD spectra demonstrated the occurrence of two Nickel peaks at 35-40 deg and 40-45 deg in Ni-M/A, but only one nickel peak at 35-40 deg in Ni-M/A-Mpro, indicating the binding of Mpro on the Nickel ions. EDS analysis reported a decrease in the concentration of Nickel on the surface of Ni-M/A from 16% to 7% when Mpro protein was loaded into the stationary phase. Importantly, our data indicated that the purity of the His-tagged protein Mpro after purification reached 97% after just one-step purification using the Ni-M/A stationary phase. Moreover, the binding capacity of Ni-M/A for Mpro was approximately 5.2 mg/g with recovery efficiency of 40%. Our results suggested Ni-M/A as a highly potential solid phase for affinity chromatography in the purification of His-tagged protein.

Solution-Phase Late-Stage Chemoselective Photocatalytic Removal of Sulfonyl and Phenacyl Groups in Peptides.

Herein, BPC catalyzed visible-light-triggered target-specific late-stage solution phase desulfonylation from tryptophan in oligopeptides is portrayed by overcoming the isolation issue up to octamers. This robust and mild method is highly predictable and chemoselective, tolerating myriad of functional groups in aza-heteroaromatics and peptides. Interestingly, reductive desulfonylation is also amenable to biologically significant reactive histidine and tyrosine side chains, signifying the versatility of the strategy. Additional efficacy of BPC is demonstrated by solution phase phenacyl deprotection from C-terminal in peptides. Furthermore, excellent catalyst loading of 0.5 mol% and recyclability demonstrate the practical utility and applicability of this strategy.

Bacterial Cellulose-Based MOF Hybrid as a Sensitive Switch Off-On Luminescent Sensor for the Selective Recognition of l-Histidine.

In this study, a stable and luminescent UiO-66-NH2 (UN) and its derivative Cu2+@UN were prepared and utilized successfully as an Off-On luminescent sensing platform for effective, selective, as well as rapid (5 min) detection of l-Histidine (l-His). The UN reveals efficient quenching in the presence of Cu2+ ions through photoinduced electron transition (PET) mechanism as a dynamic quenching process (in the range of 0.01-1 mM) forming Cu2+@UN sensing platform. However, due to the remarkable affinity between l-His and Cu2+, the luminescence of Cu2+@UN is recovered in the presence of l-His indicating Turn-On behavior via a quencher detachment mechanism (QD). A good linear relationship between the l-His concentration and luminescence intensity was observed in the range of 0.01-40 µM (R2 = 0.9978) with a detection limit of 7 nM for l-His sensing. The suggested method was successfully utilized for l-His determination in real samples with good recoveries and satisfying consequences. Moreover, the result indicates that only l-His induces a significant luminescence restoration of Cu2+@UN and that the signal is significantly greater than that of the other amino acids. Also, the portable test paper based on bacterial cellulose (BC) as the Cu2+@UNBC sensing platform was developed to conveniently evaluate the effective detection of l-His.

Edge Substitution Effects of Histidine Tautomerization Behaviors on the Structural Properties and Aggregation Properties of Aß(1-42) Mature Fibril.

Histidine behaviors play critical roles in folding and misfolding processes due to the changes in net charge and the various N/N-H orientations on imidazole rings. However, the effect of histidine tautomerization (HIE (Nε-H, ε) and HID (Nδ-H, δ) states) behaviors on the edge chain of Aß mature fibrils remains inadequately understood, which is critical for finding a strategy to disturb fibril elongation and growth. In the current study, eight independent molecular dynamics simulations were conducted to investigate such impacts on the structural and aggregation properties. Our results from three different binding models revealed that the binding contributions of edge substitution effects are primarily located between chains 1 and 2. Histidine states significantly influence the secondary structure of each domain. Further analysis confirmed that the C1_H6//C1_E11 intrachain interaction is essential in maintaining the internal stability of chain 1, while the C1_H13//C2_H13 and C1_H14//C2_H13 interchain interactions are critical in maintaining the interchain stability of the fibril structure. Our subsequent analysis revealed that the current edge substitution leads to the loss of the C1_H13//C1_E11 intrachain and C1_H13//C2_H14 interchain interactions. The N-terminal regularity was significantly directly influenced by histidine states, particularly by the residue of C1_H13. Our study provides valuable insights into the effect of histidine behaviors on the edge chain of Aß mature fibril, advancing our understanding of the histidine behavior hypothesis in misfolding diseases.

Synthesis of Amino Acid-Based Cationic Lipids and Study of the Role of the Cationic Head Group for Enhanced Drug and Nucleic Acid Delivery.

Leveraging liposomes for drug and nucleic acid delivery, though promising due to reduced toxicity and ease of preparation, faces challenges in stability and efficiency. To address this, we synthesized cationic amphiphiles from amino acids (arginine, lysine, and histidine). Histidine emerged as the superior candidate, leading to the development of three histidine-rich cationic amphiphiles for liposomes. Using the hydration method, we have prepared the liposomes and determined the optimal N/P ratios for lipoplex formation via gel electrophoresis. In vitro transfection assays compared the efficacy of our lipids to Fugene, while MTT assays gauged biocompatibility across cancer cell lines (MDA-MB 231 and MCF-7). The histidine-based lipid demonstrated marked potential in enhancing drug and nucleic acid delivery. This improvement stemmed from increased zeta potential, enhancing electrostatic interactions with nucleic acids and cellular uptake. Our findings underscore histidine's crucial role over lysine and arginine for effective delivery, revealing a significant correlation between histidine abundance and optimal performance. This study paves the way for histidine-enriched lipids as promising candidates for efficient drug and nucleic acid delivery, addressing key challenges in the field.

Effect of proline content and histidine ligation on the dynamics of Ω-loop D and the peroxidase activity of iso-1-cytochrome c.

To study how proline residues affect the dynamics of Ω-loop D (residues 70 to 85) of cytochrome c, we prepared G83P and G83A variants of yeast iso-1-cytochrome c (iso-1-Cytc) in the presence and absence of a K73H mutation. Ω-loop D is important in controlling both the electron transfer function of Cytc and the peroxidase activity of Cytc used in apoptosis because it provides the Met80 heme ligand. The G83P and G83A mutations have no effect on the global stability of iso-1-Cytc in presence or absence of the K73H mutation. However, both mutations destabilize the His73-mediated alkaline conformer relative to the native state. pH jump stopped-flow experiments show that the dynamics of the His73-mediated alkaline transition are significantly enhanced by the G83P mutation. Gated electron transfer studies show that the enhanced dynamics result from an increased rate of return to the native state, whereas the rate of loss of Met80 ligation is unchanged by the G83P mutation. Thus, the G83P substitution does not stiffen the conformation of the native state. Because bis-His heme ligation occurs when Cytc binds to cardiolipin-containing membranes, we studied the effect of His73 ligation on the peroxidase activity of Cytc, which acts as an early signal in apoptosis by causing oxygenation of cardiolipin. We find that the His73 alkaline conformer suppresses the peroxidase activity of Cytc. Thus, the bis-His ligated state of Cytc formed upon binding to cardiolipin is a negative effector for the peroxidase activity of Cytc early in apoptosis.

Histidine-assisted reduction of arylnitrenes upon photo-activation of phenyl azide chromophores in GFP-like fluorescent proteins.

The photochemically active sites of the proteins sfGFP66azF and Venus66azF, members of the green fluorescent protein (GFP) family, contain a non-canonical amino acid residue p-azidophenylalanine (azF) instead of Tyr66. The light-induced decomposition of azF at these sites leads to the formation of reactive arylnitrene (nF) intermediates followed by the formation of phenylamine-containing chromophores. We report the first study of the reaction mechanism of the reduction of the arylnitrene intermediates in sfGFP66nF and Venus66nF using molecular modeling methods. The Gibbs energy profiles for the elementary steps of the chemical reaction in sfGFP66nF are computed using molecular dynamics simulations with quantum mechanics/molecular mechanics (QM/MM) potentials. Structures and energies along the reaction pathway in Venus66nF are evaluated using a QM/MM approach. According to the results of the simulations, arylnitrene reduction is coupled with oxidation of the histidine side chain on the His148 residue located near the chromophore.

Structure prediction of physiological bis(amino acidato)copper(II) species in aqueous solution: The copper(II) compounds with l-glutamine and l-histidine.

Neutral (l-histidinato)(l-glutaminato)copper(II) [Cu(His)(Gln)] has been established as the most abundant ternary copper(II) amino acid compound of the exchangeable copper(II) pool in blood plasma. The experimental studies of Cu(His)(Gln) and bis(glutaminato)copper(II) [Cu(Gln)2] in solutions did not specify their complete geometries. To determine the geometries, this paper investigates the conformers, energy landscapes, and a structure-magnetic parameters relation of Cu(Gln)2 and Cu(His)(Gln) by the density functional theory (DFT) calculations. We assume a glycine-like coordination of Gln (other coordination patterns are dismissed because of steric reasons), and three His in-plane copper(II) binding modes. The conformational analyses are performed in the gas phase and implicitly modeled aqueous solution. The reliability of the DFT relative electronic and Gibbs free energies of the Cu(His)(Gln) conformers is confirmed by benchmarking against the corresponding energies obtained by the domain-based local pair natural orbital coupled-cluster method with singles, doubles, and perturbative triples [DLPNO-CCSD(T)]. Several cis- and trans-Cu(His)(Gln) conformers with His in the histaminate-like and glycine-like modes have low Gibbs free energies, and the greatest estimated metal-binding affinities. The DFT-calculated magnetic parameters of the low-energy conformers reproduce best the experimental electron paramagnetic resonance parameters measured in aqueous solutions for trans- and cis-Cu(Gln)2 conformers having two oxygen atoms (either from Gln or water molecules) at the apical positions, and Cu(His)(Gln) conformers having His in the histaminate-like mode with an apically placed carboxylato oxygen atom. The predicted conformational flexibility of His­copper(II)-amino acid compounds may be connected with their physiological abundance, and the role in copper(II) exchange reactions in blood plasma.

-HH and -HAAAH motifs act as fishing nets for biologically relevant metal ions in metallopeptides.

Histidine are one of the most common residues involved in transition metal ion binding in the active sites of metalloenzymes. In order to mimic enzymatic metal binding sites, it is crucial to understand the basic coordination modes of histidine residues, distributed at different positions in the peptide sequence. We show that: (i) the separation of two histidines has a large effect on complex stability - a sequence with adjusting histidine residues forms more stable complexes with Zn(II) than the one in which the residues are separated, while the contrary is observed for Cu(II) complexes, in which amide nitrogens participate in metal binding. No pronounced effect is observed for Ni(II) complexes, where the amides participate in binding at higher pH; (ii) non-coordinating amino acid residues (basic, acidic and aromatic ones) have a significant impact on complex stability; charged and aromatic residues may enhance Zn(II) binding, while the contrary is observed for the amide-binding Cu(II); (iii) cysteine containing sequences are much more effective Zn(II) and Ni(II) binding motifs at pH above 8, while histidine containing ligands are more suitable for effective Zn(II) and Ni(II) binding at lower pH.

Region-Directed Enzyme Immobilization through Engineering Protein Surface with Histidine Clusters.

Enzyme immobilization is a key enabling technology for a myriad of industrial applications, yet immobilization science is still too empirical to reach highly active and robust heterogeneous biocatalysts through a general approach. Conventional protein immobilization methods lack control over how enzymes are oriented on solid carriers, resulting in negative conformational changes that drive enzyme deactivation. Site-selective enzyme immobilization through peptide tags and protein domains addresses the orientation issue, but this approach limits the possible orientations to the N- and C-termini of the target enzyme. In this work, we engineer the surface of two model dehydrogenases to introduce histidine clusters into flexible regions not involved in catalysis, through which immobilization is driven. By varying the position and the histidine density of the clusters, we create a small library of enzyme variants to be immobilized on different carriers functionalized with different densities of various metal chelates (Co2+, Cu2+, Ni2+, and Fe3+). We first demonstrate that His-clusters can be as efficient as the conventional His-tags in immobilizing enzymes, recovering even more activity and gaining stability against some denaturing agents. Furthermore, we find that the enzyme orientation as well as the type and density of the metal chelates affect the immobilization parameters (immobilization yield and recovered activity) and the stability of the immobilized enzymes. According to proteomic studies, His-clusters enable a different enzyme orientation as compared to His-tag. Finally, these oriented heterogeneous biocatalysts are implemented in batch reactions, demonstrating that the stability achieved by an optimized orientation translates into increased operational stability.

Histidine-Based Supramolecular Nanoassembly Exhibiting Dual Enzyme-Mimetic Functions: Altering the Tautomeric Preference of Histidine to Tailor Oxidative/Hydrolytic Catalysis.

Challenges persist in replicating enzyme-like active sites with functional group arrangements in supramolecular catalysis. In this study, we present a supramolecular material comprising Fmoc-modified histidine and copper. We also investigated the impact of noncanonical amino acids (δmH and εmH), isomers of histidine, on the catalytic process. The Fmoc-δmH-based nanoassembly exhibits an approximately 15-fold increase in oxidative activity and an ∼50-fold increase in hydrolytic activity compared to Fmoc-εmH (kcat/Km). This distinction arises from differences in basicity and ligation properties between the ε- and δ-nitrogen of histidine. The addition of guanosine monophosphate further enhances the oxidative activity of the histidine- and methylated histidine-based catalysts. The Fmoc-δmH/Cu2+-based nanoassembly catalyzes the oxidation/hydrolysis cascade of 2',7'-dichlorofluorescein diacetate, benefiting from the synergistic effect between the copper center and the nonligating ε-nitrogen of histidine. These findings advance the biomimetic catalyst design and provide insights into the mechanistic role of essential residues in natural systems.

DPH1 Gene Mutations Identify a Candidate SAM Pocket in Radical Enzyme Dph1•Dph2 for Diphthamide Synthesis on EF2.

In eukaryotes, the Dph1•Dph2 dimer is a non-canonical radical SAM enzyme. Using iron-sulfur (FeS) clusters, it cleaves the cosubstrate S-adenosyl-methionine (SAM) to form a 3-amino-3-carboxy-propyl (ACP) radical for the synthesis of diphthamide. The latter decorates a histidine residue on elongation factor 2 (EF2) conserved from archaea to yeast and humans and is important for accurate mRNA translation and protein synthesis. Guided by evidence from archaeal orthologues, we searched for a putative SAM-binding pocket in Dph1•Dph2 from Saccharomyces cerevisiae. We predict an SAM-binding pocket near the FeS cluster domain that is conserved across eukaryotes in Dph1 but not Dph2. Site-directed DPH1 mutagenesis and functional characterization through assay diagnostics for the loss of diphthamide reveal that the SAM pocket is essential for synthesis of the décor on EF2 in vivo. Further evidence from structural modeling suggests particularly critical residues close to the methionine moiety of SAM. Presumably, they facilitate a geometry specific for SAM cleavage and ACP radical formation that distinguishes Dph1•Dph2 from classical radical SAM enzymes, which generate canonical 5'-deoxyadenosyl (dAdo) radicals.