Triad Motif Research Articles

Development and validation of a multiplex quantitative polymerase chain reaction assay for detecting and genotyping red sea bream iridoviral disease

Megalocytiviruses are responsible for severe mortality in various marine and freshwater fish species worldwide. Specifically, the red sea bream iridovirus (RSIV) and infectious spleen and kidney necrosis virus (ISKNV) genotypes are known to cause the red sea bream iridoviral disease (RSIVD). Although several quantitative polymerase chain reaction (qPCR) assays have been developed for diagnosing RSIVD, these methods fail to differentiate between viral genotypes within Megalocytivirus. The present study introduces an innovative multiplex qPCR assay for the precise detection of RSIVD. This diagnostic method targets the histidine triad motif (HIT)-like protein and ORF080L genes, which are unique markers for the RSIV and ISKNV genotypes. The assay exhibited high analytical sensitivity, expressed as the 95% limit of detection (LoD95%), of 8.69 copies/5 μL for RSIV and 19.07 copies/5 μL for ISKNV. Notably, the assay exhibited no cross-reactivity and was unaffected by common inhibitory substances. The diagnostic sensitivity and specificity were 100% for both RSIV and ISKNV, demonstrating the reliability of the assay. The multiplex qPCR assay was 100–1000 times more sensitive than conventional PCR methods, emphasizing its importance in early-stage infection detection. The repeatability and reproducibility of the assay were confirmed through extensive testing, showing low variation and high correlation across different technicians and laboratory settings. Furthermore, the ability of the assay to selectively detect and differentiate between RSIVD genotypes represents a significant advancement in aquatic animal disease diagnostics. This multiplex qPCR assay offers a rapid, specific, sensitive, and quantitative diagnostic tool for global RSIVD surveillance efforts and contributes to the prevention and understanding of disease progression in the aquaculture industry.

Complete degradation of di-n-butyl phthalate by Glutamicibacter sp. strain 0426 with a novel pathway.

Di-n-butyl phthalate (DBP) is widely used as plasticizer that has potential carcinogenic, teratogenic, and endocrine effects. In the present study, an efficient DBP-degrading bacterial strain 0426 was isolated and identified as a Glutamicibacter sp. strain 0426. It can utilize DBP as the sole source of carbon and energy and completely degraded 300mg/L of DBP within 12h. The optimal conditions (pH 6.9 and 31.7°C) for DBP degradation were determined by response surface methodology and DBP degradation well fitted with the first-order kinetics. Bioaugmentation of contaminated soil with strain 0426 enhanced DBP (1mg/g soil) degradation, indicating the application potential of strain 0426 for environment DBP removal. Strain 0426 harbors a distinctive DBP hydrolysis mechanism with two parallel benzoate metabolic pathways, which may account for the remarkable performance of DBP degradation. Sequences alignment has shown that an alpha/beta fold hydrolase (WP_083586847.1) contained a conserved catalytic triad and pentapeptide motif (GX1SX2G), of which function is similar to phthalic acid ester (PAEs) hydrolases and lipases that can efficiently catalyze hydrolysis of water-insoluble substrates. Furthermore, phthalic acid was converted to benzoate by decarboxylation, which entered into two different pathways: one is the protocatechuic acid pathway under the role of pca cluster, and the other is the catechol pathway. This study demonstrates a novel DBP degradation pathway, which broadens our understanding of the mechanisms of PAE biodegradation.

Characterization of a Malabaricane-Type Triterpene Synthase from Astragalus membranaceus and Enzymatic Synthesis of Astramalabaricosides.

Triterpenoids are a large and medicinally important group of natural products with a wide range of biological and pharmacological effects. Among them, malabaricane-type triterpenoids are a rare group of terpenoids with a 6,6,5-tricyclic ring system, and a few malabaricane triterpene synthases have been characterized to date. Here, an arabidiol synthase AmAS for the formation of the malabaricane-type 6,6,5-tricyclic triterpenoid skeleton in astramalabaricosides biosynthesis was characterized from Astragalus membranaceus. Multiple sequence alignment, site-directed mutagenesis, and molecular docking of AmAS reveal that residues Q256 and Y258 are essential for AmAS activity, and the triad motif IIH725-727 was the critical residue necessary for its product specificity. Mutation of IIH725-727 with VFN led to the formation of seven tricyclic, tetracyclic, and pentacyclic triterpenoids (1-7). Glycosylation of malabaricane-type triterpenoids in the biosynthesis of astramalabaricosides was also explored. Three triterpenoids (1, 5, and 6) displayed potent inhibitory effects against influenza A virus in vitro. These findings provide insights into malabaricane-type triterpenoids biosynthesis in A. membranaceus and access to diverse bioactive triterpenoids for drug discovery.

Rational identification of a catalytically promiscuous nitrilase by predicting a unique catalytic triad motif feature through an in silico strategy

A key motif of a catalytically promiscuous nitrilase (CP-NLase) was identified by mimicking active site features from cyanide hydratases (CHTs) in combination with structural analysis.

Cloning, overexpression, purification, characterization and structural modelling of a metabolically active Fe2+ dependent 2,6-dichloro-p-hydroquinone 1,2-dioxygenase (CpsA) from Bacillus cereus strain AOA-CPS_1

2,6-Dichloro-p-hydroquinone (DiCHQ) aromatic-ring cleavage by DiCHQ 1,2-dioxygenase (CpsA) is very crucial for complete transformation of pentachlorophenol (PCP) to 2-chloromaleylacetate in Bacillus cereus AOA-CPS_1 (BcAOA). The 978 bp gene (cpsA) was detected and amplified in the genome of BcAOA; cloned, overexpressed and purified to homogeneity. CpsA showed a single ≅36.9 kDa protein band on SDS-PAGE and exhibited optimum activity at 30 °C and pH 9.0. CpsA was stable between 20 °C and 40 °C, and also retained about 90% of its activity at 60 °C for 120 min. The enzyme retained about 90% activity between pH 9.0 and 11.5 and 60% activity at pH 13.0. CpsA was found to be Fe2+ dependent as about 90% increased activity was observed in the presence of FeSO4. CpsA showed apparent vmax, Km, kcat and kcat/Km of 27.77 ± 0.9 μMs−1, 0.990 ± 0.03 mM, 4.20 ± 0.04 s−1 and 4.24 ± 0.03 s−1 mM−1, respectively at pH 9.0. Analysis of the reaction products via GC–MS confirmed 2-chloromaleylacetate as the ring-cleavage product. CpsA 3D structure revealed a conserved 2-His-1-carboxylate facial triad motif (His 9, His 244 and Thr 11), with Fe3+ at the centre. Findings from this study provide new insights into the involvement of this enzyme in PCP degradation and suggests alternate possible mechanism of ring-cleavage by dioxygenases.

Characterisation of a lysophospholipase from Lactobacillus mucosae.

In this study, we characterised a novel lysophospholipase (LysoPL) from the L. mucosae LM1 strain. The gene, LM-lysoPL, encoding LysoPL from L. mucosae LM1 was cloned, analyzed, and expressed. LM-lysoPL contained a conserved region and catalytic triad motif responsible for lysophospholipase activity. After purification, UHPLC-MS analysis showed that recombinant LM-LysoPL hydrolyzed phosphatidic acid, generating lysophosphatidic acid. The enzyme had greater hydrolytic activity against C16 and C18 fatty acids, indicating a preference for long-chain fatty acids. Enzymatic assays showed that the optimal pH and temperature of recombinant LM-LysoPL were 7 and 30 °C, respectively, and it was enzymatically active within a narrow pH range. To the best of our knowledge, this is the first study to identify and characterize a lysophospholipase from lactic acid bacteria. Our findings provide a basis for understanding the probiotic role of L. mucosae LM1 in the gut.

Crystal Structure of Histidine Triad Nucleotide-Binding Protein from the Pathogenic Fungus Candida albicans.

Histidine triad nucleotide-binding protein (HINT) is a member of the histidine triad (HIT) superfamily, which has hydrolase activity owing to a histidine triad motif. The HIT superfamily can be divided to five classes with functions in galactose metabolism, DNA repair, and tumor suppression. HINTs are highly conserved from archaea to humans and function as tumor suppressors, translation regulators, and neuropathy inhibitors. Although the structures of HINT proteins from various species have been reported, limited structural information is available for fungal species. Here, to elucidate the structural features and functional diversity of HINTs, we determined the crystal structure of HINT from the pathogenic fungus Candida albicans (CaHINT) in complex with zinc ions at a resolution of 2.5 Å. Based on structural comparisons, the monomer of CaHINT overlaid best with HINT protein from the protozoal species Leishmania major. Additionally, structural comparisons with human HINT revealed an additional helix at the C-terminus of CaHINT. Interestingly, the extended C-terminal helix interacted with the N-terminal loop (α1–β1) and with the α3 helix, which appeared to stabilize the dimerization of CaHINT. In the C-terminal region, structural and sequence comparisons showed strong relationships among 19 diverse species from archea to humans, suggesting early separation in the course of evolution. Further studies are required to address the functional significance of variations in the C-terminal region. This structural analysis of CaHINT provided important insights into the molecular aspects of evolution within the HIT superfamily.

Structural characterisation of the HT3 motif of the polyhistidine triad protein D from Streptococcus pneumoniae.

The bacterium Streptococcus pneumoniae (the pneumococcus) is a major human pathogen that requires Zn2+ for its survival and virulence in the host environment. Polyhistidine triad protein D (PhtD) has a known role in pneumococcal Zn2+ homeostasis. However, the mechanistic basis of PhtD function remains unclear, partly due to a lack of structural information. Here, we determined the crystal structure of the fragment PhtD269-339 , containing the third Zn2+ -binding histidine triad (HT) motif of the protein. Analysis of the structure suggests that Zn2+ binding occurs at the surface of the protein and that all five HT motifs in the protein bind Zn2+ and share similar structures. These new structural insights aid in our understanding of how the Pht proteins facilitate pneumococcal Zn2+ acquisition.

Metal-chelating 3-hydroxypyrimidine-2,4-diones inhibit human cytomegalovirus pUL89 endonuclease activity and virus replication

Human cytomegalovirus terminase complex cleaves the concatemeric genomic viral DNA into unit lengths during genome packaging and particle assembly. Terminase complex ATPase and endonuclease activity is provided by the viral protein pUL89. pUL89 is an attractive drug target because its activities are required for infectious virus production. A domain located in the C-terminus of pUL89 has an RNase H/integrase-like fold and endonuclease activity that can be inhibited by compounds featuring a chelating triad motif. Previously, we developed a novel ELISA approach to screen for pUL89 inhibitors. In this report, we used the ELISA to identify 3-hydroxypyrimidine-2,4-dione as a promising scaffold for pUL89 inhibitor development. Several potent pUL89 inhibitors yielded low micromolar IC50 values in the enzymatic assay and low micromolar EC50 values for inhibition of HCMV replication. Two representative compounds inhibitory effects depended upon metal ions and occurred late in virus replication consistent with pUL89 inhibitors in infected cells.

Alpha/beta-hydrolases: A unique structural motif coordinates catalytic acid residue in 40 protein fold families.

The alpha/beta-hydrolases are a family of acid-base-nucleophile catalytic triad enzymes with a common fold, but using a wide variety of substrates, having different pH optima, catalyzing unique catalytic reactions and often showing improved chemical and thermo stability. The ABH enzymes are prime targets for protein engineering. Here, we have classified active sites from 51 representative members of 40 structural ABH fold families into eight distinct conserved geometries. We demonstrate the occurrence of a common structural motif, the catalytic acid zone, at the catalytic triad acid turn. We show that binding of an external ligand does not change the structure of the catalytic acid zone and both the ligand-free and ligand-bound forms of the protein belong to the same catalytic acid zone subgroup. We also show that the catalytic acid zone coordinates the position of the catalytic histidine loop directly above its plane, and consequently, fixes the catalytic histidine in a proper position near the catalytic acid. Finally, we demonstrate that the catalytic acid zone plays a key role in multi-subunit complex formation in ABH enzymes, and is involved in interactions with other proteins. As a result, we speculate that each of the catalytic triad residues has its own supporting structural scaffold, similar to the catalytic acid zone, described above, which together form the extended catalytic triad motif. Each scaffold coordinates the function of its respective catalytic residue, and can even compensate for the loss of protein function, if the catalytic amino acid is mutated.

Synthesis of part structures of Cryptococcus neoformans serotype C capsular polysaccharide

Cryptococcus neoformans is a fungal pathogen that can cause life-threatening infections in immunocompromised patients. The development of a vaccine based on the capsular polysaccharide of C. neoformans is still an open challenge due to the heterogeneity of the capsular polysaccharide and the difficulty of identifying protective epitopes. Therefore, construction of structurally defined part structures of the C. neoformans GXM capsule is in great demand. Herein is presented the synthesis of a 3-O-naphthalenylmethyl protected trisaccharide thioglycoside building block which is present in C. neoformans serotype C polysaccharide. Its property as a donor in a glycosylation reaction with a model acceptor has been evaluated together with its behaviour as an acceptor following removal of the temporary protecting group. The heavily branched hexasaccharide was obtained in good yields and excellent α-selectivity. The frame shifted octasaccharide structural triad motif for serotype C was also prepared following the same building block strategy. For the first time this structural motif, which is the most substituted amongst the four C. neoformans serotypes, was prepared. Three synthesized C. neoformans serotype C fragments of varying size, from penta-up to octasaccharide, were deprotected and will be included in unique glycoarrays to further investigate the possibility to develop a synthetic vaccine against this pathogen.

Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model.

Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic efficiencies towards target reactions.

Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants

Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity.

Crystal structure of HINT from Helicobacter pylori.

Proteins belonging to the histidine triad (HIT) superfamily bind nucleotides and use the histidine triad motif to carry out dinucleotidyl hydrolase, nucleotidyltransferase and phosphoramidite hydrolase activities. Five different branches of this superfamily are known to exist. Defects in these proteins in humans are linked to many diseases such as ataxia, diseases of RNA metabolism and cell-cycle regulation, and various types of cancer. The histidine triad nucleotide protein (HINT) is nearly identical to proteins that have been classified as protein kinase C-interacting proteins (PKCIs), which also have the ability to bind and inhibit protein kinase C. The structure of HINT, which exists as a homodimer, is highly conserved from humans to bacteria and shares homology with the product of fragile histidine triad protein (FHit), a tumour suppressor gene of this superfamily. Here, the structure of HINT from Helicobacter pylori (HpHINT) in complex with AMP is reported at a resolution of 3 Å. The final model has R and Rfree values of 26 and 28%, respectively, with good electron density. Structural comparison with previously reported homologues and phylogenetic analysis shows H. pylori HINT to be the smallest among them, and suggests that it branched out separately during the course of evolution. Overall, this structure has contributed to a better understanding of this protein across the animal kingdom.

ARGONAUTE SUBFAMILY GENES IN THE SMALL BROWN PLANTHOPPER, Laodelphax striatellus (HEMIPTERA: DELPHACIDAE).

Argonaute (AGO) proteins are essential catalytic components of the RNA-induced silencing complex and play central roles in RNA interference. Using a combination of bioinformatics and rapid amplification of cDNA ends (RACE) methods, putative AGO subfamily members, ls-AGO1 and ls-AGO2, were cloned and characterized from the small brown planthopper, Laodelphax striatellus. The open reading frame (ORF) of ls-AGO1 is 2,820 bp long, encoding a putative protein of 939 amino acid residues, and ls-AGO2 contains an ORF of 2,490 bp, encoding 829 amino acid residues. The expected conserved PAZ and PIWI domains, and the conserved Asp-Asp-His (DDH) catalytic triad motif in the PIWI domain were observed in both ls-AGO1 and ls-AGO2. Reverse transcription-qPCR (RT-qPCR) results showed that both ls-AGO1 and ls-AGO2 were expressed in all developmental stages of L. striatellus with highest mRNA abundance in eggs. Expression of ls-AGO1 and ls-AGO2 was significantly decreased in adult insects in response to acquisition of rice black-streaked dwarf virus by second instar nymphs. mRNA expression of ls-AGO1 was significantly downregulated in response to low and high temperatures, but expression of ls-AGO2 was only affected by low temperature. ls-AGO1 and ls-AGO2 were initially downregulated when insects were transferred from rice to maize and to the wild grass Brachypodium distachyon, but expression showed partial or complete recovery 7 days after transfer. These results document that AGO subfamily members of L. striatellus are ubiquitously expressed at different developmental stages and respond to various stresses. Thus, AGO subfamily may act in regulating the stress-response of L. striatellus by controlling related gene expression.

Designing potent antimicrobial peptides by disulphide linked dimerization and N-terminal lipidation to increase antimicrobial activity and membrane perturbation: Structural insights into lipopolysaccharide binding

HypothesisThe remarkable rise in multi-drug resistant Gram-negative bacterial pathogens is a major concern to the well being of humans as well as susceptible plants. In recent years, diseases associated with inflammation and septicemia have already become a global health issue. Therefore, there is a rising demand for the development of novel “super” antibiotics. In this context, antimicrobial peptides offer an attractive, alternate therapeutic solution to conventional antibiotics. ExperimentsMicrobroth dilution assay was performed to investigate the antimicrobial activities of the two designed peptides against Gram negative bacterial pathogens. Fluorescence studies including NPN dye uptake assay, Calcein entrapped vesicle leakage assay, quenching and anisotropy in presence of lipopolysaccharide (LPS) were performed to elucidate binding interactions and enhanced membrane permeabilisation. Hemolytic assay and endotoxin/LPS neutralisation assay were performed to study the hemolytic effects and LPS scavenging abilities of the peptides. High resolution NMR studies were performed to obtain insights into LPS-peptide interaction at the molecular level. FindingsHere, we report more potent analogues of previously designed peptide VG16KRKP, designed through dimerization via Cys–Cys disulphide linkage and N-terminal lipidation. Similar to the parent peptide, VG16KRKP, the modified analogue peptides are non hemolytic in nature, but possessed, 2–10-fold increase in antibacterial activities against E. coli, human pathogen Pseudomonas aeruginosa and the devastating plant pathogen, Xanthomonas campestris pv. campestris as well as membrane permeabilization, and endotoxin neutralization. LPS bound solution structure of both analogues, as determined by NMR spectroscopy, reveal that the conserved hydrophobic triad motif, formed by Trp5, Leu11 and Phe12 is compactly organized and stabilized either by the acyl chain or disulphide bond. This structural constraint accounts for the separation of polar face from the hydrophobic face of the peptides. Our novel peptides designed through Cys–Cys dimerization and N-terminal lipidation, will serve as a template to develop more potent antimicrobials in future, to control plant and human diseases.

Growing scale-free networks with tunable distributions of triad motifs

Network motifs are local structural patterns and elementary functional units of complex networks in real world, which can have significant impacts on the global behavior of these systems. Many models are able to reproduce complex networks mimicking a series of global features of real systems, however the local features such as motifs in real networks have not been well represented. We propose a model to grow scale-free networks with tunable motif distributions through a combined operation of preferential attachment and triad motif seeding steps. Numerical experiments show that the constructed networks have adjustable distributions of the local triad motifs, meanwhile preserving the global features of power-law distributions of node degree, short average path lengths of nodes, and highly clustered structures.

Roles of Ala-149 in the catalytic activity of diadenosine tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv

Diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap4A) phosphorylase from Mycobacterium tuberculosis H37Rv (MtAPA) belongs to the histidine triad motif (HIT) superfamily, but is the only member with an alanine residue at position 149 (Ala-149). Enzymatic analysis revealed that the Ala-149 deletion mutant displayed substrate specificity for diadenosine 5',5'''-P(1),P(5)-pentaphosphate and was inactive on Ap4A and other substrates that are utilized by the wild-type enzyme.

Death Effector Domain Flexibility in Mediating Protein-Protein Interactions

PED/PEA-15 (phosphoprotein enriched in diabetes/astrocytes, 15 kD) is a small, non-catalytic, death-effector domain (DED) containing protein, that is widely expressed in different tissues and highly conserved among mammals. PED/PEA-15 has been found to interact with several protein targets in various pathways, including FADD and procaspase-8 (apoptosis), ERK1/2 (cell cycle entry), and PLD1/2 (diabetes). We have previously reported a surprising conformational change of PED/PEA-15 DED upon interaction with ERK2 using NMR dynamics and residual dipolar coupling (RDC) data. In the complex, PED/PEA-15 utilizes helices α1, α5, and α6 of the DED, in addition to the C-terminal tail, to binding to ERK2, while helices α2, α3, and α4 are highly flexible in the complex, and adopt a distinct relative orientation comparing to the free-form conformation. We have additionally modeled the PED/PEA-15 conformations in the ERK2 complex using CS/RDC-Rosetta protocol. Based on our NMR model of PED/PEA-15 complex with ERK2, we propose that the conserved charge triad motif on DED surface, comprising of D19-R72-D74L, which is located at a hinge position between these two dynamically distinct segments, mediates the necessary conformational changes to accommodate ERK2 binding. We believe that various conformations of the DED, facilitated by large number of surface polar interactions, may attribute to its ability to interact with structurally and functionally diverse proteins.

Tuning Reactivity and Mechanism in Oxidation Reactions by Mononuclear Nonheme Iron(IV)-Oxo Complexes

Mononuclear nonheme iron enzymes generate high-valent iron(IV)-oxo intermediates that effect metabolically important oxidative transformations in the catalytic cycle of dioxygen activation. In 2003, researchers first spectroscopically characterized a mononuclear nonheme iron(IV)-oxo intermediate in the reaction of taurine: α-ketogultarate dioxygenase (TauD). This nonheme iron enzyme with an iron active center was coordinated to a 2-His-1- carboxylate facial triad motif. In the same year, researchers obtained the first crystal structure of a mononuclear nonheme iron(IV)-oxo complex bearing a macrocyclic supporting ligand, [(TMC)Fe(IV)(O)](2+) (TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecene), in studies that mimicked the biological enzymes. With these breakthrough results, many other studies have examined mononuclear nonheme iron(IV)-oxo intermediates trapped in enzymatic reactions or synthesized in biomimetic reactions. Over the past decade, researchers in the fields of biological, bioinorganic, and oxidation chemistry have extensively investigated the structure, spectroscopy, and reactivity of nonheme iron(IV)-oxo species, leading to a wealth of information from these enzymatic and biomimetic studies. This Account summarizes the reactivity and mechanisms of synthetic mononuclear nonheme iron(IV)-oxo complexes in oxidation reactions and examines factors that modulate their reactivities and change their reaction mechanisms. We focus on several reactions including the oxidation of organic and inorganic compounds, electron transfer, and oxygen atom exchange with water by synthetic mononuclear nonheme iron(IV)-oxo complexes. In addition, we recently observed that the C-H bond activation by nonheme iron(IV)-oxo and other nonheme metal(IV)-oxo complexes does not follow the H-atom abstraction/oxygen-rebound mechanism, which has been well-established in heme systems. The structural and electronic effects of supporting ligands on the oxidizing power of iron(IV)-oxo complexes are significant in these reactions. However, the difference in spin states between nonheme iron(IV)-oxo complexes with an octahedral geometry (with an S = 1 intermediate-spin state) or a trigonal bipyramidal (TBP) geometry (with an S = 2 high-spin state) does not lead to a significant change in reactivity in biomimetic systems. Thus, the importance of the high-spin state of iron(IV)-oxo species in nonheme iron enzymes remains unexplained. We also discuss how the axial and equatorial ligands and binding of redox-inactive metal ions and protons to the iron-oxo moiety influence the reactivities of the nonheme iron(IV)-oxo complexes. We emphasize how these changes can enhance the oxidizing power of nonheme metal(IV)-oxo complexes in oxygen atom transfer and electron-transfer reactions remarkably. This Account demonstrates great advancements in the understanding of the chemistry of mononuclear nonheme iron(IV)-oxo intermediates within the last 10 years.