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α-Ketoglutarate-dependent non-heme iron (α-KG NHI) oxygenases compose one of the largest superfamilies of tailoring enzymes that play key roles in structural and functional diversifications. During the biosynthesis of meroterpenoids, α-KG NHI oxygenases catalyze diverse types of chemical reactions, including hydroxylation, desaturation, epoxidation, endoperoxidation, ring-cleavage, and skeletal rearrangements. Due to their catalytic versatility, keen attention has been focused on functional analyses of α-KG NHI oxygenases. This chapter provides detailed methodologies for the functional analysis of the fungal α-KG NHI oxygenase SptF, which plays an important role in the structural diversification of andiconin-derived meroterpenoids. The procedures included describe how to prepare the meroterpenoid substrate using a heterologous fungal host, measure the in vitro enzymatic activity of SptF, and how to perform structural and mutagenesis studies on SptF. These protocols are also applicable to functional analyses of other α-KG NHI oxygenases.

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Rieske non-heme iron oxygenases are ubiquitously expressed in prokaryotes. These enzymes catalyze a wide variety of reactions, including cis-dihydroxylation, mono-hydroxylation, sulfoxidation, and demethylation. They contain a Rieske-type [2Fe-2S] cluster and an active site with a mono-nuclear iron bound to a 2-His carboxylate triad. Naphthalene 1,2 dioxygenase, a representative of this family, catalyzes the conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. This transformation requires naphthalene, two electrons, and an oxygen molecule. The first structure of the terminal oxygenase component of a Rieske non-heme iron oxygenase to be determined was naphthalene 1,2 dioxygenase (NDO-O). In this article, we describe in detail the methods used to recombinantly express and purify NDO-O in rich and minimal salts media, the crystallization of NDO-O for structure determination by X-ray crystallography, the challenges faced, and the methods used for the preparation of enzyme ligand complexes. The methods used here resulted in the determination of several NDO-O complexes with aromatic substrates, nitric oxide, oxygen molecule, and products, leading to an initial understanding of the mechanism of enzyme catalysis and the molecular determinants of the regio- and stereo-specificity of this class of enzymes.

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Oxazinomycin is a C-nucleoside natural product characterized by a 1,3-oxazine ring linked to ribose via a C-C glycosidic bond. Construction of the 1,3-oxazine ring depends on the activity of OzmD, which is a mononuclear non-heme iron-dependent enzyme from a family of enzymes that contain a domain of unknown function (DUF) 4243. OzmD catalyzes an unusual oxidative ring rearrangement of a pyridine derivative that releases cyanide as a by-product in the final stage of oxazinomycin biosynthesis. The intrinsic sensitivity of the OzmD substrate to oxygen along with the oxygen dependency of catalysis presents significant challenges in conducting in vitro enzymatic assays. This chapter describes the detailed procedures that have been used to characterize OzmD, including protein preparation, activity assays, and reaction by-product identification.

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Extradiol dioxygenases are a class of non-heme iron-dependent enzymes found in eukaryotes and prokaryotes that play a vital role in the aerobic catabolism of aromatic compounds. They are generally divided into three evolutionarily independent superfamilies with different protein folds. Our recent studies have shed light on the catalytic mechanisms and structure-function relationships of two specific extradiol dioxygenases: 3-hydroxyanthranilate-3,4-dioxygenase, a Type III enzyme essential in mammals for producing a precursor for nicotinamide adenine dinucleotide, and L-3,4-dihydroxyphenylalanine dioxygenase, an uncommon form of Type I enzymes involved in natural product biosynthesis. This work details the expression and isolation methods for these extradiol dioxygenases and introduces approaches to achieve homogeneity and high occupancy of the enzyme metal centers. Techniques such as ultraviolet-visible and electron paramagnetic resonance spectroscopies, as well as oxygen electrode measurements, are discussed for probing the interaction of the non-heme iron center with ligands and characterizing enzymatic activities. Moreover, protein crystallization has been demonstrated as a powerful tool to study these enzymes. We highlight in crystallo reactions and single-crystal spectroscopic methods to further elucidate enzymatic functions and protein dynamics.

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Cyclopropane and azacyclopropane, also known as aziridine, moieties are found in natural products. These moieties serve as pivotal components that lead to a broad spectrum of biological activities. While diverse strategies involving various classes of enzymes are utilized to catalyze formation of these strained three-membered rings, how non-heme iron and 2-oxoglutarate (Fe/2OG) dependent enzymes enable regio- and stereo-selective C-C and C-N ring closure has only been reported very recently. Herein, we present detailed experimental protocols for mechanistically studying Fe/2OG enzymes that catalyze cyclopropanation and aziridination reactions. These protocols include protein purification, in vitro assays, biophysical spectroscopies, and isotope-tracer experiments. We also report how to use in silico approaches to look for Fe/2OG aziridinases. Furthermore, our current mechanistic understanding of three-membered ring formation is discussed. These results not only shed light on the reaction mechanisms of Fe/2OG enzymes-catalyzed cyclopropanation and aziridination, but also open avenues for expanding the reaction repertoire of the Fe/2OG enzyme superfamily.

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The aromatic amino acid hydroxylases phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase utilize a non-heme iron to catalyze the hydroxylation of the aromatic rings of their amino acid substrates, with a tetrahydropterin serving as the source of the electrons necessary for the monooxygenation reaction. These enzymes have been subjected to a variety of biochemical and biophysical approaches, resulting in a detailed understanding of their structures and mechanism. We summarize here the experimental approaches that have led to this understanding.

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Mammalian cysteamine dioxygenase (ADO), a mononuclear non-heme Fe(II) enzyme with three histidine ligands, plays a key role in cysteamine catabolism and regulation of the N-degron signaling pathway. Despite its importance, the catalytic mechanism of ADO remains elusive. Here, we describe an HPLC-MS assay for characterizing thiol dioxygenase catalytic activities and a metal-substitution approach for mechanistic investigation using human ADO as a model. Two proposed mechanisms for ADO differ in oxygen activation: one involving a high-valent ferryl-oxo intermediate. We hypothesized that substituting iron with a metal that has a disfavored tendency to form high-valent states would discriminate between mechanisms. This chapter details the expression, purification, preparation, and characterization of cobalt-substituted ADO. The new HPLC-MS assay precisely measures enzymatic activity, revealing retained reactivity in the cobalt-substituted enzyme. The results obtained favor the concurrent dioxygen transfer mechanism in ADO. This combined approach provides a powerful tool for studying other non-heme iron thiol oxidizing enzymes.

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Determination of substrate binding affinity (Kd) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the Kd of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis coupled with High Performance Liquid Chromatography (HPLC) for subsequent detection. This method can be performed in anaerobic glove bag settings. It requires readily available HPLC instrumentation for ligand quantitation and is adaptable to meet the needs of a variety of substrate affinity measurements.

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Rieske non-heme iron oxygenases (ROs) possess the ability to catalyze a wide range of reactions. Their ability to degrade aromatic compounds is a unique characteristic and makes ROs interesting for a variety of potential applications. However, purified ROs can be challenging to work with due to low stability and long, complex electron transport chains. Whole cell biocatalysis represents a quick and reliable method for characterizing the activity of ROs and harnessing their metabolic potential. In this protocol, we outline a step-by-step protocol for the overexpression of ROs for whole cell biocatalysis and characterization. We have utilized a caffeine-degrading, N-demethylation system, expressing the RO genes ndmA and ndmD, as an example of this method.

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Flavodiiron NO reductases (FNORs) are important enzymes in microbial pathogenesis, as they equip microbes with resistance to the human immune defense agent nitric oxide (NO). Despite much efforts, intermediates that would provide insight into how the non-heme diiron active sites of FNORs reduce NO to N2O could not be identified. Computations predict that iron-hyponitrite complexes are the key species, leading from NO to N2O. However, the coordination chemistry of non-heme iron centers with hyponitrite is largely unknown. In this study, we report the reactivity of two non-heme iron complexes with preformed hyponitrite. In the case of [Fe(TPA)(CH3CN)2](OTf)2, cleavage of hyponitrite and formation of an Fe2(NO)2 diamond core is observed. With less Lewis-acidic [Fe2(BMPA-PhO)2(OTf)2] (2), reaction with Na2N2O2 in polar aprotic solvent leads to the formation of a red complex, 3. X-ray crystallography shows that 3 is a tetranuclear iron-hyponitrite complex, [{Fe2(BMPA-PhO)2}2(µ-N2O2)](OTf)2, with a unique hyponitrite binding mode. This species provided the unique opportunity to us to study the interaction of hyponitrite with non-heme iron centers and the reactivity of the bound hyponitrite ligand. Here, either protonation or oxidation of 3 is found to induce N2O formation, supporting the hypothesis that hyponitrite is a viable intermediate in NO reduction.

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Non-heme iron enzymes play key roles in antibiotic, neurotransmitter, and natural product biosynthesis, DNA repair, hypoxia regulation, and disease states. These enzymes had been refractory to traditional bioinorganic spectroscopic methods. Thus, we developed variable-temperature variable-field magnetic circular dichroism (VTVH MCD) spectroscopy to experimentally define the excited and ground ligand field states of non-heme ferrous enzymes (Solomon et al., 1995). This method provides detailed geometric and electronic structure insight and thus enables a molecular level understanding of catalytic mechanisms. Application of this method across the five classes of non-heme ferrous enzymes has defined that a general mechanistic strategy is utilized where O2 activation is controlled to occur only in the presence of all cosubstrates.

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Microbial metabolism is an important driving force for the elimination of 4-chlorophenoxyacetic acid residues in the environment. The α-Ketoglutarate-dependent dioxygenase (TfdA) or 2,4-D oxygenase (CadAB) catalyzes the cleavage of the aryl ether bond of 4-chlorophenoxyacetic acid to 4-chlorophenol, which is one of the important pathways for the initial metabolism of 4-chlorophenoxyacetic acid by microorganisms. However, strain Cupriavidus sp. DL-D2 could utilize 4-chlorophenoxyacetic acid but not 4-chlorophenol for growth. This scarcely studied degradation pathway may involve novel enzymes that has not yet been characterized. Here, a gene cluster (designated cpd) responsible for the catabolism of 4-chlorophenoxyacetic acid in strain DL-D2 was cloned and identified, and the dioxygenase CpdA/CpdB responsible for the initial degradation of 4-chlorophenoxyacetic acid was successfully expressed, which could catalyze the conversion of 4-chlorphenoxyacetic acid to 4-chlorocatechol. Then, an aromatic cleavage enzyme CpdC further converts 4-chlorocatechol into 3-chloromuconate. The results of substrate degradation experiments showed that CpdA/CpdB could also degrade 3-chlorophenoxyacetic acid and phenoxyacetic acid, and homologous cpd gene clusters were widely discovered in microbial genomes. Our findings revealed a novel degradation mechanism of 4-chlorophenoxyacetic acid at the molecular level.

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Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O2) concentrations. Here, we have reprogrammed cellular hypoxia (low O2) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non-heme iron dependent O2 sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O2 to PHD2's catalytic core. We show that systematic modification of these residues can open the constriction topology of PHD2's gas tunnel. Using kinetic stopped-flow measurements with NO as a surrogate diatomic gas, we demonstrate up to 3.5-fold enhancement in its association rate to the iron center of tunnel-engineered mutants. Our most effectively designed mutant displays 9-fold enhanced catalytic efficiency (kcat/KM = 830 ± 40 M-1 s-1) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF-1α, as compared to WT PHD2 (kcat/KM = 90 ± 9 M-1 s-1). Furthermore, transfection of plasmids that express designed PHD2 mutants in HEK-293T mammalian cells reveal significant reduction of HIF-1α and downstream hypoxia response transcripts under hypoxic conditions of 1% O2. Overall, these studies highlight activation of PHD2 as a new pathway to reprogram hypoxia responses and HIF signaling in cells.

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Non-heme iron-dependent sulfoxide/selenoxide synthases (NHISS) constitute a unique metalloenzyme class capable of installing a C-S/Se bond onto histidine to generate thio/selenoimidazole antioxidants, such as ergothioneine and ovothiol. These natural products are increasingly recognized for their health benefits. Among associated ergothioneine-biosynthetic enzymes, type IV EgtBs stand out, as they exhibit low sequence similarity with other EgtB subfamilies due to their recent divergence from the ovothiol-biosynthetic enzyme OvoA. Herein, we present crystal structures of two representative EgtB-IV enzymes, offering insights into the basis for this evolutionary convergence and enhancing our understanding of NHISS active site organization more broadly. The ability to interpret how key residues modulate substrate specificity and regioselectivity has implications for downstream identification of divergent reactivity within the NHISS family. To this end, we identify a previously unclassified clade of OvoA-like enzymes with a seemingly hybrid set of characteristics, suggesting they may represent an evolutionary intermediate between OvoA and EgtB-IV.

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Highly selective C-H functionalization remains an ongoing challenge in organic synthetic methodologies. Biocatalysts are robust tools for achieving these difficult chemical transformations. Biocatalyst engineering has often required directed evolution or structure-based rational design campaigns to improve their activities. In recent years, machine learning has been integrated into these workflows to improve the discovery of beneficial enzyme variants. In this work, we combine a structure-based machine-learning algorithm with classical molecular dynamics simulations to down select mutations for rational design of a non-heme iron-dependent lysine dioxygenase, LDO. This approach consistently resulted in functional LDO mutants and circumvents the need for extensive study of mutational activity before-hand. Our rationally designed single mutants purified with up to 2-fold higher yields than WT and displayed higher total turnover numbers (TTN). Combining five such single mutations into a pentamutant variant, LPNYI LDO, leads to a 40% improvement in the TTN (218±3) as compared to WT LDO (TTN = 160±2). Overall, this work offers a low-barrier approach for those seeking to synergize machine learning algorithms with pre-existing protein engineering strategies.

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PURPOSE: Poor bioavailability may contribute to iron deficiency among children in high-resource countries, but iron bioavailability of Australian pre-schooler diets is unknown. This study aimed to estimate the bioavailability of Australian pre-schooler iron intakes across the day and by eating occasions to identify optimal timing for intervention, by using five previously developed algorithms, and to estimate the proportion of children with intakes of absorbable iron below the requirements. METHODS: Dietary data of children aged 2 to < 6 y (n = 812) from the 2011-12 National Nutrition and Physical Activity Survey were collected via two 24-h recalls. Usual food and nutrient intakes were estimated via Multiple Source Method. Phytate, polyphenol, and heme iron values were sourced from international databases or the literature. Five previously published algorithms were applied to observed dietary data to estimate iron bioavailability and calculate the prevalence of children with intakes of absorbable iron below requirements. RESULTS: Pre-schooler daily iron bioavailability was low (2.7-10.5%) and corresponded to intakes of 0.18-0.75 mg/d of absorbable iron. The proportion of children with inadequate intakes of absorbable iron ranged between 32 and 98%. For all eating occasions, dinner offered iron of the greatest bioavailability (4.2-16.4%), while iron consumed at breakfast was of the lowest bioavailability (1.2-5.6%). CONCLUSION: Future strategies are required to improve intakes of bioavailable iron for pre-schoolers to prevent the risk of deficiency. These strategies could include the encouragement of concomitant consumption of enhancers of iron absorption with iron-rich sources, particularly at breakfast.

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Protein engineering is crucial to improve enzymes' efficiency and robustness for industrial biocatalysis. NOV1 is a bacterial dioxygenase that holds biotechnological potential by catalyzing the one-step oxidation of the lignin-derived isoeugenol into vanillin, a popular flavoring agent used in food, cleaning products, cosmetics and pharmaceuticals. This study aims to enhance NOV1 activity and operational stability through the identification of distal hotspots, located at more than 9 Šfrom the active site using Zymspot, a tool that predicts advantageous distant mutations, streamlining protein engineering. A total of 41 variants were constructed using site-directed mutagenesis and the six most active enzyme variants were then recombined. Two variants, with two and three mutations, showed nearly a 10-fold increase in activity and up to 40-fold higher operational stability than the wild-type. Furthermore, these variants show 90-100 % immobilization efficiency in metal affinity resins, compared to approximately 60 % for the wild-type. In bioconversions where 50 mM of isoeugenol was added stepwise over 24-h cycles, the 1D2 variant produced approximately 144 mM of vanillin after six reaction cycles, corresponding to around 22 mg, indicating a 35 % molar conversion yield. This output was around 2.5 times higher than that obtained using the wild-type. Our findings highlight the efficacy of distal protein engineering in enhancing enzyme functions like activity, stability, and metal binding selectivity, thereby fulfilling the criteria for industrial biocatalysts. This study provides a novel approach to enzyme optimization that could have significant implications for various biotechnological applications.

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Free interconversion of cytochrome C (CytC) between native ferrous (Cyt-FeII) and oxidized ferric (CytC-FeIII) states is necessary to maintain the respiratory function of mitochondria. Disturbances in CytC-FeIII to total CytC ratio may indicate mitochondrial dysfunction and apoptosis. Thus, tracking CytC oxidation state delivers important information about cellular physiology. In this work, we propose a novel methodology based on resonance Raman (rR) imaging optimized uniquely to track and qualitatively analyze the transition of Cyt-FeII to CytC-FeIII within live cells without affecting their morphology. None of the commonly used excitation lines allows such clear-cut differentiation, contrary to the 405 nm applied in this work. The presented methodology provides a novel pathway in the label-free detection of ferrous and ferric heme proteins.

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DosT and DosS are heme-based kinases involved in sensing and signaling O2 tension in the microenvironment of Mycobacterium tuberculosis (Mtb). Under conditions of low O2, they activate >50 dormancy-related genes and play a pivotal role in the induction of dormancy and associated drug resistance during tuberculosis infection. In this work, we reexamine the O2 binding affinities of DosT and DosS to show that their equilibrium dissociation constants are 3.3±1.0 µM and 0.46±0.08 µM respectively, which are six to eight-fold stronger than what has been widely referred to in literature. Furthermore, stopped-flow kinetic studies reveal association and dissociation rate constants of 0.84 µM-1 s-1 and 2.8 s-1, respectively for DosT, and 7.2 µM-1 s-1 and 3.3 s-1, respectively for DosS. Remarkably, these tighter O2 binding constants correlate with distinct stages of hypoxia-induced non-replicating persistence in the Wayne model of Mtb. This knowledge opens doors to deconvoluting the intricate interplay between hypoxia adaptation stages and the signal transduction capabilities of these important heme-based O2 sensors.

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The color, lipid oxidation, heme iron (HI) and non-heme iron (NHI) contents, metmyoglobin content and Soret band of myoglobin of ground pork subjected to supercritical CO2 treatment under different conditions, or to heat treatment (40°C, 2 h) and subsequent storage at 4°C were evaluated during 9-day period. Supercritical CO2 treatment significantly increased CIE L* and CIE b* values of ground pork during subsequent storage, while the HI content was slightly affected. In general, CIE a* value and metmyoglobin content were decreased. Supercritical CO2 treatment for 2 h could increase the thiobarbituric acid-reactive substances (TBARS) value, while treatment for 1 h or less had no effect. The NHI content could be increased only after treatment at above 40°C or 17.2 MPa for 2 h. The Soret band of myoglobin was shifted to longer wavelength. Increasing treatment temperature from 35°C to 45°C could increase CIE L*, CIE a*, CIE b* and TBARS values, HI and NHI contents of the ground pork, while decreasing metmyoglobin content. As the treatment pressure increased from 13.8 MPa to 20.7 MPa, CIE b* and TBARS values were decreased, while the NHI and metmyoglobin contents were increased. However, the other parameters were unchanged. Extending exposure time from 0.5 h to 2 h could increase CIE L*, CIE b* and TBARS values, HI contents, while decreasing CIE a* value and metmyoglobin content. Correlation analysis showed that the TBARS value was significantly and negatively correlated with the HI content or metmyoglobin content in samples treated at 40°C or above for 2 h.