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AIM: This study aims to investigate the key elements for successful operation and management of primary otolaryngologic clinics in Taiwan amidst a declining birth rate and increasing competition among clinics. It employs the Innovation Through Tradition (ITT) theory as a theoretical framework to develop an operational model for effective management strategies. METHODS: This research utilized the triangulation method to identify key elements crucial for the operation and management of primary otolaryngologic clinics. Five key elements were identified, namely service attitude, medication efficacy, diagnostic and treatment procedures, treatment costs, and operating hours. Outpatient satisfaction was analyzed using Donabedian's structure-process-outcomes model to assess the impact of these elements on patient experience. RESULTS: Analysis revealed that service attitude significantly influences outpatient visits, indicating its paramount importance in clinic management. Patient satisfaction was highest in the service outcome dimension, emphasizing the significance of effective treatment outcomes. However, satisfaction was lowest in the service structure dimension, indicating potential areas for improvement in clinic infrastructure and organization. CONCLUSION: Understanding these key elements and enhancing outpatient satisfaction can drive improvements in the quality of medical services, contributing to the overall success of primary otolaryngologic clinics.
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Hyoscyamine 6ß-hydroxylase (H6H) is an iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase that produces the prolifically administered antinausea drug, scopolamine. After its namesake hydroxylation reaction, H6H then couples the newly installed C6 oxygen to C7 to produce the drug's epoxide functionality. Oxoiron(IV) (ferryl) intermediates initiate both reactions by cleaving C-H bonds, but it remains unclear how the enzyme switches the target site and promotes (C6)O-C7 coupling in preference to C7 hydroxylation in the second step. In one possible epoxidation mechanism, the C6 oxygen wouldâanalogously to mechanisms proposed for the Fe/2OG halogenases and, in our more recent study, N-acetylnorloline synthase (LolO)âcoordinate as alkoxide to the C7-H-cleaving ferryl intermediate to enable alkoxyl coupling to the ensuing C7 radical. Here, we provide structural and kinetic evidence that H6H does not employ substrate coordination or repositioning for the epoxidation step but instead exploits the distinct spatial dependencies of competitive C-H cleavage (C6 vs C7) and C-O-coupling (oxygen rebound vs cyclization) steps to promote the two-step sequence. Structural comparisons of ferryl-mimicking vanadyl complexes of wild-type H6H and a variant that preferentially 7-hydroxylates instead of epoxidizing 6ß-hydroxyhyoscyamine suggest that a modest (â¼10°) shift in the Fe-O-H(C7) approach angle is sufficient to change the outcome. The 7-hydroxylation:epoxidation partition ratios of both proteins increase more than 5-fold in 2H2O, reflecting an epoxidation-specific requirement for cleavage of the alcohol O-H bond, which, unlike in the LolO oxacyclization, is not accomplished by iron coordination in advance of C-H cleavage.
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A number of bacteria are known to produce isonitrile-containing peptides (INPs) that facilitate metal transport and are important for cell survival; however, considerable structural variation is observed among INPs depending on the producing organism. While non-heme iron 2-oxoglutarate dependent isonitrilases catalyze isonitrile formation, how the natural variation in INP structure is controlled and its implications for INP bioactivity remain open questions. Herein, total chemical synthesis is utilized with X-Ray crystallographic analysis of mycobacterial isonitrilases to provide a structural model of substrate specificity that explains the longer alkyl chains observed in mycobacterial versus Streptomyces INPs. Moreover, proton NMR titration experiments demonstrate that INPs regardless of alkyl chain length are specific for binding copper instead of zinc. These results suggest that isonitrilases may act as gatekeepers in modulating the observed biological distribution of INP structures and this distribution may be primarily related to differing metal transport requirements among the producing strains.
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Non-heme mononuclear iron dependent (NHM-Fe) enzymes exhibit exceedingly diverse catalytic reactivities. Despite their catalytic versatilities, the mononuclear iron centers in these enzymes show a relatively simple architecture, in which an iron atom is ligated with 2-4 amino acid residues, including histidine, aspartic or glutamic acid. In the past two decades, a common high-valent reactive iron intermediate, the S=2 oxyferryl (Fe(IV)-oxo or Fe(IV)=O) species, has been repeatedly discovered in NHM-Fe enzymes containing a 2-His-Fe or 2-His-1-carboxylate-Fe center. However, for 3-His/4-His-Fe enzymes, no common reactive intermediate has been identified. Recently, we have spectroscopically characterized the first S=1 Fe(IV) intermediate in a 3-His-Fe containing enzyme, OvoA, which catalyzes a novel oxidative carbon-sulfur bond formation. In this review, we summarize the broad reactivities demonstrated by S=2 Fe(IV)-oxo intermediates, the discovery of the first S=1 Fe(IV) intermediate in OvoA and the mechanistic implication of such a discovery, and the intrinsic reactivity differences of the S=2 and the S=1 Fe(IV)-oxo species. Finally, we postulate the possible reasons to utilize an S=1 Fe(IV) species in OvoA and their implications to other 3-His/4-His-Fe enzymes.
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Oral cancer ranks fourth among malignancies among Taiwanese men and is the eighth most common cancer among men worldwide in terms of general diagnosis. The purpose of the current study was to investigate how low-density lipoprotein receptor-related protein 1B (LDL receptor related protein 1B; LRP1B) gene polymorphisms affect oral squamous cell carcinoma (OSCC) risk and progression in individuals with diabetes mellitus (DM). Three LRP1B single-nucleotide polymorphisms (SNPs), including rs10496915, rs431809, and rs6742944, were evaluated in 311 OSCC cases and 300 controls. Between the case and control groups, we found no evidence of a significant correlation between the risk of OSCC and any of the three specific SNPs. Nevertheless, in evaluating the clinicopathological criteria, individuals with DM who possess a minimum of one minor allele of rs10496915 (AC + CC; p = 0.046) were significantly associated with tumor size compared with those with homozygous major alleles (AA). Similarly, compared to genotypes homologous for the main allele (GG), rs6742944 genotypes (GA + AA; p = 0.010) were more likely to develop lymph node metastases. The tongue and the rs6742944 genotypes (GA + AA) exhibited higher rates of advanced clinical stages (p = 0.024) and lymph node metastases (p = 0.007) when compared to homozygous alleles (GG). LRP1B genetic polymorphisms appear to be prognostic and diagnostic markers for OSCC and DM, as well as contributing to genetic profiling research for personalized medicine.
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Carcinoma de Células Escamosas , Diabetes Mellitus , Neoplasias de Cabeza y Cuello , Neoplasias de la Boca , Masculino , Humanos , Neoplasias de la Boca/genética , Metástasis Linfática , Carcinoma de Células Escamosas/genética , Polimorfismo de Nucleótido Simple , Carcinoma de Células Escamosas de Cabeza y Cuello , Receptores de LDL/genéticaRESUMEN
Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F420. Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F420 by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L-glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.
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Ácido Fólico , Ácido Glutámico , Humanos , Péptido Sintasas/metabolismo , Bacterias/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
CT scanning of the chest is one the most important imaging modalities available for pulmonary disease diagnosis. Lung segmentation plays a crucial step in the pipeline of computer-aided analysis and diagnosis. As deep learning models have achieved human-level accuracy in semantic segmentation of anatomical structures, we propose to use trained deep learning models to predict both healthy and infectious areas in chest CT slices. The semantic segmentation results are summarized and visualized using volume rendering technology in the form of roadmaps. The roadmaps consist of both location and volume information that can be used as a location guidance for inspecting suspected pulmonary lesions of chest CT and can possibly be combined into a rapid triage algorithm for treating acute pulmonary diseases.Clinical Relevance- This research applied trained semantic segmentation models in identifying normal lung and pneumonic infection areas to generate a roadmap for assisting medical doctors in browsing chest CT and prognostication.
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Neumonía , Humanos , Neumonía/diagnóstico por imagen , Tomografía Computarizada por Rayos X/métodos , Pulmón/patología , Tórax , AlgoritmosRESUMEN
Chlamydia protein associating with death domains (CADD), the founding member of a recently discovered class of nonheme dimetal enzymes termed hemeoxygenase-like dimetaloxidases (HDOs), plays an indispensable role in pathogen survival. CADD orchestrates the biosynthesis of p-aminobenzoic acid (pABA) for integration into folate via the self-sacrificial excision of a protein-derived tyrosine (Tyr27) and several additional processing steps, the nature and timing of which have yet to be fully clarified. Nuclear magnetic resonance (NMR) and proteomics approaches reveal the source and probable timing of amine installation by a neighboring lysine (Lys152). Turnover studies using limiting O2 have identified a para-aminobenzaldehyde (pABCHO) metabolic intermediate that is formed on the path to pABA formation. The use of pABCHO and other probe substrates shows that the heterobimetallic Fe/Mn form of the enzyme is capable of oxygen insertion to generate the pABA-carboxylate.
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Ácido 4-Aminobenzoico , para-Aminobenzoatos , para-Aminobenzoatos/metabolismo , Ácido 4-Aminobenzoico/metabolismo , Ácido Fólico/metabolismoRESUMEN
BelL and HrmJ are α-ketoglutarate-dependent nonheme iron enzymes that catalyze the oxidative cyclization of 6-nitronorleucine, resulting in the formation of two diastereomeric 3-(2-nitrocyclopropyl)alanine (Ncpa) products containing trans-cyclopropane rings with (1'R,2'R) and (1'S,2'S) configurations, respectively. Herein, we investigate the catalytic mechanism and stereodivergency of the cyclopropanases. The results suggest that the nitroalkane moiety of the substrate is first deprotonated to produce the nitronate form. Spectroscopic analyses and biochemical assays with substrates and analogues indicate that an iron(IV)-oxo species abstracts proS-H from C4 to initiate intramolecular C-C bond formation. A hydroxylation intermediate is unlikely to be involved in the cyclopropanation reaction. Additionally, a genome mining approach is employed to discover new homologues that perform the cyclopropanation of 6-nitronorleucine to generate cis-configured Ncpa products with (1'R,2'S) or (1'S,2'R) stereochemistries. Sequence and structure comparisons of these cyclopropanases enable us to determine the amino acid residues critical for controlling the stereoselectivity of cyclopropanation.
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Aminocaproatos , Estereoisomerismo , Oxidación-ReducciónRESUMEN
The cytochrome P450 (CYP) AspB is involved in the biosynthesis of the diketopiperazine (DKP) aspergilazine A. Tryptophan-linked dimeric DKP alkaloids are a large family of natural products that are found in numerous species and exhibit broad and often potent bioactivity. The proposed mechanisms for C-N bond formation by AspB, and similar C-C bond formations by related CYPs, have invoked the use of a ferryl-intermediate as an oxidant to promote substrate dimerization. Here, the parallel application of steady-state and transient kinetic approaches reveals a very different mechanism that involves a ferric-superoxide species as a primary oxidant to initiate DKP-assembly. Single turnover kinetic isotope effects and a substrate analog suggest the probable nature and site for abstraction. The direct observation of CYP-superoxide reactivity rationalizes the atypical outcome of AspB and reveals a new reaction manifold in heme enzymes.
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Hierro , Superóxidos , Dimerización , Sistema Enzimático del Citocromo P-450 , Oxidantes , Dicetopiperazinas , Dipéptidos , Electrólitos , CatálisisRESUMEN
Mononuclear nonheme iron(II) and 2-oxoglutarate (Fe/2OG)-dependent oxygenases and halogenases are known to catalyze a diverse set of oxidative reactions, including hydroxylation, halogenation, epoxidation, and desaturation in primary metabolism and natural product maturation. However, their use in abiotic transformations has mainly been limited to C-H oxidation. Herein, we show that various enzymes of this family, when reconstituted with Fe(II) or Fe(III), can catalyze Mukaiyama hydration-a redox neutral transformation. Distinct from the native reactions of the Fe/2OG enzymes, wherein oxygen atom transfer (OAT) catalyzed by an iron-oxo species is involved, this nonnative transformation proceeds through a hydrogen atom transfer (HAT) pathway in a 2OG-independent manner. Additionally, in contrast to conventional inorganic catalysts, wherein a dinuclear iron species is responsible for HAT, the Fe/2OG enzymes exploit a mononuclear iron center to support this reaction. Collectively, our work demonstrates that Fe/2OG enzymes have utility in catalysis beyond the current scope of catalytic oxidation.
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Hierro , Oxigenasas , Oxigenasas/metabolismo , Hierro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxidación-Reducción , Catálisis , HidrógenoRESUMEN
Ergothioneine (ESH) and ovothiol A (OSHA) are two natural thiol-histidine derivatives. ESH has been implicated as a longevity vitamin and OSHA inhibits the proliferation of hepatocarcinoma. The key biosynthetic step of ESH and OSHA in the aerobic pathways is the O2 -dependent C-S bond formation catalyzed by non-heme iron enzymes (e.g., OvoA in ovothiol biosynthesis), but due to the lack of identification of key reactive intermediate the mechanism of this novel reaction is unresolved. In this study, we report the identification and characterization of a kinetically competent S=1 iron(IV) intermediate supported by a four-histidine ligand environment (three from the protein residues and one from the substrate) in enabling C-S bond formation in OvoA from Methyloversatilis thermotoleran, which represents the first experimentally observed intermediate spin iron(IV) species in non-heme iron enzymes. Results reported in this study thus set the stage to further dissect the mechanism of enzymatic oxidative C-S bond formation in the OSHA biosynthesis pathway. They also afford new opportunities to study the structure-function relationship of high-valent iron intermediates supported by a histidine rich ligand environment.
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Histidina , Hierro , Histidina/metabolismo , Ligandos , Catálisis , Estrés OxidativoRESUMEN
Aziridines are compounds with a nitrogen-containing three-membered ring. When it is incorporated into natural products, the reactivity of the strained ring often drives the biological activities of aziridines. Despite its importance, the enzymes and biosynthetic strategies deployed to install this reactive moiety remain understudied. Herein, we report the use of in silico methods to identify enzymes with potential aziridine-installing (aziridinase) functionality. To validate candidates, we reconstitute enzymatic activity in vitro and demonstrate that an iron(IV)-oxo species initiates aziridine ring closure by the C-H bond cleavage. Furthermore, we divert the reaction pathway from aziridination to hydroxylation using mechanistic probes. This observation, isotope tracing experiments using H218O and 18O2, and quantitative product analysis, provide evidence for the polar capture of a carbocation species by the amine in the pathway to aziridine installation.
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Aziridinas , Hierro , Hierro/química , Hidroxilación , CatálisisRESUMEN
Chlamydia protein associating with death domains (CADD) is involved in the biosynthesis of para-aminobenzoate (pABA), an essential component of the folate cofactor that is required for the survival and proliferation of the human pathogen Chlamydia trachomatis. The pathway used by Chlamydiae for pABA synthesis differs from the canonical multi-enzyme pathway used by most bacteria that relies on chorismate as a metabolic precursor. Rather, recent work showed pABA formation by CADD derives from l-tyrosine. As a member of the emerging superfamily of heme oxygenase-like diiron oxidases (HDOs), CADD was proposed to use a diiron cofactor for catalysis. However, we report maximal pABA formation by CADD occurs upon the addition of both iron and manganese, which implicates a heterobimetallic Fe:Mn cluster is the catalytically active form. Isotopic labeling experiments and proteomics studies show that CADD generates pABA from a protein-derived tyrosine (Tyr27), a residue that is â¼14 Å from the dimetal site. We propose that this self-sacrificial reaction occurs through O2 activation by a probable Fe:Mn cluster through a radical relay mechanism that connects to the "substrate" Tyr, followed by amination and direct oxygen insertion. These results provide the molecular basis for pABA formation in C. trachomatis, which will inform the design of novel therapeutics.
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Proteínas Bacterianas , Chlamydia trachomatis , Oxigenasas , Tirosina , para-Aminobenzoatos , Proteínas Bacterianas/metabolismo , Chlamydia trachomatis/enzimología , Ácido Fólico , Hierro/metabolismo , Manganeso/metabolismo , Oxígeno/metabolismo , Oxigenasas/metabolismo , Tirosina/metabolismo , para-Aminobenzoatos/metabolismoRESUMEN
Two different types of desaturations are employed by iron- and 2-oxoglutarate-dependent (Fe/2OG) enzymes to construct vinyl isonitrile and isocyanoacrylate moieties found in isonitrile-containing natural products. A substrate-bound protein structure reveals a plausible strategy to affect desaturation and hints at substrate promiscuity of these enzymes. Analogs are synthesized and used as mechanistic probes to validate structural observations. Instead of proceeding through hydroxylated intermediate as previously proposed, a plausible carbocation species is utilized to trigger C=C bond installation. These Fe/2OG enzymes can also accommodate analogs with opposite chirality and different functional groups including isonitrile-(D)-tyrosine, N-formyl tyrosine, and phloretic acid, while maintaining the reaction selectivity.
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Hierro , Ácidos Cetoglutáricos , Hierro/metabolismo , Ácidos Cetoglutáricos/metabolismo , TirosinaRESUMEN
Despite the diversity of reactions catalyzed by 2-oxoglutarate-dependent nonheme iron (Fe/2OG) enzymes identified in recent years, only a limited number of these enzymes have been investigated in mechanistic detail. In particular, several Fe/2OG-dependent enzymes capable of catalyzing isocyanide formation have been reported. While the glycine moiety has been identified as a biosynthon for the isocyanide group, how the actual conversion is effected remains obscure. To elucidate the catalytic mechanism, we characterized two previously unidentified (AecA and AmcA) along with two known (ScoE and SfaA) Fe/2OG-dependent enzymes that catalyze N≡C triple bond installation using synthesized substrate analogues and potential intermediates. Our results indicate that isocyanide formation likely entails a two-step sequence involving an imine intermediate that undergoes decarboxylation-assisted desaturation to yield the isocyanide product. Results obtained from the in vitro experiments are further supported by mutagenesis, the product-bound enzyme structure, and in silico analysis.
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Utilization of mononuclear iron- and 2-oxoglutarate-dependent (Fe/2OG) enzymes to enable C-H bond functionalization is a widely used strategy to diversify the structural complexity of natural products. Besides those well-studied reactions including hydroxylation, epoxidation, and halogenation, in the biosynthetic pathway of dehydrofosmidomycin, an Fe/2OG enzyme is reported to catalyze desaturation, alkyl chain elongation, along with demethylation in which trimethyl-2-aminoethylphosphonate is converted into methyldehydrofosmidomycin. How this transformation takes place is largely unknown. Herein, we characterized the reactive species, revealed the structure of the reaction intermediate, and used mechanistic probes to investigate the reaction pathway and mechanism. These results led to the elucidation of a two-step process in which the first reaction employs a long-lived Fe(IV)-oxo species to trigger CâC bond installation. During the second reaction, the olefin installed in situ enables C-C bond formation that is accompanied with a C-N bond cleavage and hydroxylation to furnish the alkyl chain elongation and demethylation. This work expands the reaction repertoire of Fe/2OG enzymes by introducing a new pathway to the known C-C bond formation mechanisms utilized by metalloenzymes.
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Hierro , Ácidos Cetoglutáricos , Alquenos/química , Catálisis , Hidroxilación , Hierro/química , Ácidos Cetoglutáricos/metabolismoRESUMEN
Hormaomycins and belactosins are peptide natural products that contain unusual cyclopropane moieties. Bioinformatics analysis of the corresponding biosynthetic gene clusters showed that two conserved genes, hrmI/belK and hrmJ/belL, were potential candidates for catalyzing cyclopropanation. Using in vivo and in vitro assays, the functions of HrmI/BelK and HrmJ/BelL were established. HrmI and BelK, which are heme oxygenase-like dinuclear iron enzymes, catalyze oxidation of the ϵ-amino group of l-lysine to afford l-6-nitronorleucine. Subsequently, HrmJ and BelL, which are iron- and α-ketoglutarate-dependent oxygenases, effectively convert l-6-nitronorleucine into 3-(trans-2-nitrocyclopropyl)-alanine through C4-C6 bond installation. These observations disclose a novel pathway of cyclopropane ring construction and exemplify the new chemistry involving metalloenzymes in natural product biosynthesis.
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Ciclopropanos/metabolismo , Depsipéptidos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metaloproteínas/metabolismo , Catálisis , Ciclopropanos/química , Depsipéptidos/química , Péptidos y Proteínas de Señalización Intercelular/química , Metaloproteínas/química , Estructura MolecularRESUMEN
Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS's catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel-Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs.
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Berberidaceae/enzimología , Medicamentos Herbarios Chinos/química , Ligasas/química , Proteínas de Plantas/química , Podofilotoxina/análogos & derivados , Oxidación-Reducción , Podofilotoxina/químicaRESUMEN
BesC catalyzes the iron- and O2-dependent cleavage of 4-chloro-l-lysine to form 4-chloro-l-allylglycine, formaldehyde, and ammonia. This process is a critical step for a biosynthetic pathway that generates a terminal alkyne amino acid which can be leveraged as a useful bio-orthogonal handle for protein labeling. As a member of an emerging family of diiron enzymes that are typified by their heme oxygenase-like fold and a very similar set of coordinating ligands, recently termed HDOs, BesC performs an unusual type of carbon-carbon cleavage reaction that is a significant departure from reactions catalyzed by canonical dinuclear-iron enzymes. Here, we show that BesC activates O2 in a substrate-gated manner to generate a diferric-peroxo intermediate. Examination of the reactivity of the peroxo intermediate with a series of lysine derivatives demonstrates that BesC initiates this unique reaction trajectory via cleavage of the C4-H bond; this process represents the rate-limiting step in a single turnover reaction. The observed reactivity of BesC represents the first example of a dinuclear-iron enzyme that utilizes a diferric-peroxo intermediate to capably cleave a C-H bond as part of its native function, thus circumventing the formation of a high-valent intermediate more commonly associated with substrate monooxygenations.