Radical-relay C(sp3)-H azidation catalyzed by an engineered nonheme iron enzyme.

Nonheme iron enzymes are versatile biocatalysts for a broad range of unique and powerful transformations, such as hydroxylation, chlorination, and epimerization as well as cyclization/ring-opening of organic molecules. Beyond their native biological functions, these enzymes are robust for engineering due to their structural diversity and high evolvability. Based on enzyme promiscuity and directed evolution as well as inspired by synthetic organic chemistry, nonheme iron enzymes can be repurposed to catalyze reactions previously only accessible with synthetic catalysts. To this end, our group has engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for new-to-nature radical transformations. In particular, we have demonstrated that a nonheme iron enzyme, (4-hydroxyphenyl)pyruvate dioxygenase from streptomyces avermitilis (SavHppD), can be repurposed to enable abiological radical-relay process to access C(sp3)-H azidation products. This represents the first known instance of enzymatic radical relay azidation reactions. In this chapter, we describe the detailed experimental protocol to convert promiscuous nonheme iron enzymes into efficient and selective biocatalyst for radical relay azidation reactions. One round of directed evolution is described in detail, which includes the generation and handling of site-saturation mutagenesis, protein expression and whole-cell reactions screening in a 96-well plate. These protocol details might be useful to engineer various nonheme iron enzymes for other applications.

Functional analysis of an α-ketoglutarate-dependent non-heme iron oxygenase in fungal meroterpenoid biosynthesis.

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.

Expression, purification and characterization of non-heme iron-dependent mono-oxygenase OzmD in oxazinomycin biosynthesis.

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.

Radical fluorine transfer catalysed by an engineered nonheme iron enzyme.

Nonheme iron enzymes stand out as one of the most versatile biocatalysts for molecular functionalization. They facilitate a wide array of chemical transformations within biological processes, including hydroxylation, chlorination, epimerization, desaturation, cyclization, and more. Beyond their native biological functions, these enzymes possess substantial potential as powerful biocatalytic platforms for achieving abiological metal-catalyzed reactions, owing to their functional and structural diversity and high evolvability. To this end, our group has recently engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for abiological radical transformations not previously known in biology. Notably, we have demonstrated that a nonheme iron enzyme, (S)-2-hydroxypropylphosphonate epoxidase from Streptomyces viridochromogenes (SvHppE), can be repurposed into an efficient and selective biocatalyst for radical fluorine transfer reactions. This marks the first known instance of a redox enzymatic process for C(sp3)F bond formation. This chapter outlines the detailed experimental protocol for engineering SvHPPE for fluorination reactions. Furthermore, the provided protocol could serve as a general guideline that might facilitate other engineering endeavors targeting nonheme iron enzymes for novel catalytic functions.

The NADH recycling enzymes TsaC and TsaD regenerate reducing equivalents for Rieske oxygenase chemistry.

Many microorganisms use both biological and nonbiological molecules as sources of carbon and energy. This resourcefulness means that some microorganisms have mechanisms to assimilate pollutants found in the environment. One such organism is Comamonas testosteroni, which metabolizes 4-methylbenzenesulfonate and 4-methylbenzoate using the TsaMBCD pathway. TsaM is a Rieske oxygenase, which in concert with the reductase TsaB consumes a molar equivalent of NADH. Following this step, the annotated short-chain dehydrogenase/reductase and aldehyde dehydrogenase enzymes TsaC and TsaD each regenerate a molar equivalent of NADH. This co-occurrence ameliorates the need for stoichiometric addition of reducing equivalents and thus represents an attractive strategy for integration of Rieske oxygenase chemistry into biocatalytic applications. Therefore, in this work, to overcome the lack of information regarding NADH recycling enzymes that function in partnership with Rieske non-heme iron oxygenases (Rieske oxygenases), we solved the X-ray crystal structure of TsaC to a resolution of 2.18 Å. Using this structure, a series of substrate analog and protein variant combination reactions, and differential scanning fluorimetry experiments, we identified active site features involved in binding NAD+ and controlling substrate specificity. Further in vitro enzyme cascade experiments demonstrated the efficient TsaC- and TsaD-mediated regeneration of NADH to support Rieske oxygenase chemistry. Finally, through in-depth bioinformatic analyses, we illustrate the widespread co-occurrence of Rieske oxygenases with TsaC-like enzymes. This work thus demonstrates the utility of these NADH recycling enzymes and identifies a library of short-chain dehydrogenase/reductase enzyme prospects that can be used in Rieske oxygenase pathways for in situ regeneration of NADH.

Heterodimeric Non-heme Iron Enzymes in Fungal Meroterpenoid Biosynthesis.

Talaromyolides (1-6) are a group of unusual 6/6/6/6/6/6 hexacyclic meroterpenoids with (3R)-6-hydroxymellein and 4,5-seco-drimane substructures, isolated from the marine fungus Talaromyces purpureogenus. We have identified the biosynthetic gene cluster tlxA-J by heterologous expression in Aspergillus, in vitro enzyme assays, and CRISPR-Cas9-based gene inactivation. Remarkably, the heterodimer of non-heme iron (NHI) enzymes, TlxJ-TlxI, catalyzes three steps of oxidation including a key reaction, hydroxylation at C-5 and C-9 of 12, the intermediate with 3-ketohydroxydrimane scaffold, to facilitate a retro-aldol reaction, leading to the construction of the 4,5-secodrimane skeleton and characteristic ketal scaffold of 1-6. The products of TlxJ-TlxI, 1 and 4, were further hydroxylated at C-4'ß by another NHI heterodimer, TlxA-TlxC, and acetylated by TlxB to yield the final products, 3 and 6. The X-ray structural analysis coupled with site-directed mutagenesis provided insights into the heterodimer TlxJ-TlxI formation and its catalysis. This is the first report to show that two NHI proteins form a heterodimer for catalysis and utilizes a novel methodology to create functional oxygenase structures in secondary metabolite biosynthesis.

Maternal Iron Deficiency Modulates Placental Transcriptome and Proteome in Mid-Gestation of Mouse Pregnancy.

BACKGROUND: Maternal iron deficiency (ID) is associated with poor pregnancy and fetal outcomes. The effect is thought to be mediated by the placenta but there is no comprehensive assessment of placental responses to maternal ID. Additionally, whether the influence of maternal ID on the placenta differs by fetal sex is unknown. OBJECTIVES: To identify gene and protein signatures of ID mouse placentas at mid-gestation. A secondary objective was to profile the expression of iron genes in mouse placentas across gestation. METHODS: We used a real-time PCR-based array to determine the mRNA expression of all known iron genes in mouse placentas at embryonic day (E) 12.5, E14.5, E16.5, and E19.5 (n = 3 placentas/time point). To determine the effect of maternal ID, we performed RNA sequencing and proteomics in male and female placentas from ID and iron-adequate mice at E12.5 (n = 8 dams/diet). RESULTS: In female placentas, 6 genes, including transferrin receptor (Tfrc) and solute carrier family 11 member 2, were significantly changed by maternal ID. An additional 154 genes were altered in male ID placentas. A proteomic analysis quantified 7662 proteins in the placenta. Proteins translated from iron-responsive element (IRE)-containing mRNA were altered in abundance; ferritin and ferroportin 1 decreased, while TFRC increased in ID placentas. Less than 4% of the significantly altered genes in ID placentas occurred both at the transcriptional and translational levels. CONCLUSIONS: Our data demonstrate that the impact of maternal ID on placental gene expression in mice is limited in scope and magnitude at mid-gestation. We provide strong evidence for IRE-based transcriptional and translational coordination of iron gene expression in the mouse placenta. Finally, we discover sexually dimorphic effects of maternal ID on placental gene expression, with more genes and pathways altered in male compared with female mouse placentas.

Identification of the electron donor to flavodiiron proteins in Synechocystis sp. PCC 6803 by in vivo spectroscopy.

Flavodiiron proteins (FDPs) of photosynthetic organisms play a photoprotective role by reducing oxygen to water and thus avoiding the accumulation of excess electrons on the photosystem I (PSI) acceptor side under stress conditions. In Synechocystis sp. PCC 6803 grown under high CO2, both FDPs Flv1 and Flv3 are indispensable for oxygen reduction. We performed a detailed in vivo kinetic study of wild-type (WT) and Δflv1/3 strains of Synechocystis using light-induced NADPH fluorescence and near-infrared absorption of iron-sulfur clusters from ferredoxin and the PSI acceptors (FAFB), collectively named FeS. These measurements were performed under conditions where the Calvin-Benson cycle is inactive or poorly activated. Under such conditions, the NADPH decay following a short illumination decays in parallel in both strains and exhibits a time lag which is correlated to the presence of reduced FeS. On the contrary, reduced FeS decays much faster in WT than in Δflv1/3 (13 vs 2 s-1). These data unambiguously show that reduced ferredoxin, or possibly reduced FAFB, is the direct electron donor to the Flv1/Flv3 heterodimer. Evidences for large reduction of (FAFB) and recombination reactions within PSI were also provided by near-infrared absorption. Mutants lacking either the NDH1-L complex, the homolog of complex I of respiration, or the Pgr5 protein show no difference with WT in the oxidation of reduced FeS following a short illumination. These observations question the participation of a significant cyclic electron flow in cyanobacteria during the first seconds of the induction phase of photosynthesis.

Structural and mechanistic aspects of carotenoid cleavage dioxygenases (CCDs).

Carotenoid cleavage dioxygenases (CCDs) comprise a superfamily of mononuclear non-heme iron proteins that catalyze the oxygenolytic fission of alkene bonds in carotenoids to generate apocarotenoid products. Some of these enzymes exhibit additional activities such as carbon skeleton rearrangement and trans-cis isomerization. The group also includes a subfamily of enzymes that split the interphenyl alkene bond in molecules such as resveratrol and lignostilbene. CCDs are involved in numerous biological processes ranging from production of light-sensing chromophores to degradation of lignin derivatives in pulping waste sludge. These enzymes exhibit unique features that distinguish them from other families of non-heme iron enzymes. The distinctive properties and biological importance of CCDs have stimulated interest in their modes of catalysis. Recent structural, spectroscopic, and computational studies have helped clarify mechanistic aspects of CCD catalysis. Here, we review these findings emphasizing common and unique properties of CCDs that enable their variable substrate specificity and regioselectivity. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.

Evolution-guided engineering of non-heme iron enzymes involved in nogalamycin biosynthesis.

Microbes are competent chemists that are able to generate thousands of chemically complex natural products with potent biological activities. The key to the formation of this chemical diversity has been the rapid evolution of secondary metabolism. Many enzymes residing on these metabolic pathways have acquired atypical catalytic properties in comparison with their counterparts found in primary metabolism. The biosynthetic pathway of the anthracycline nogalamycin contains two such proteins, SnoK and SnoN, belonging to nonheme iron and 2-oxoglutarate-dependent mono-oxygenases. In spite of structural similarity, the two proteins catalyze distinct chemical reactions; SnoK is a C2-C5″ carbocyclase, whereas SnoN catalyzes stereoinversion at the adjacent C4″ position. Here, we have identified four structural regions involved in the functional differentiation and generated 30 chimeric enzymes to probe catalysis. Our analyses indicate that the carbocyclase SnoK is the ancestral form of the enzyme from which SnoN has evolved to catalyze stereoinversion at the neighboring carbon. The critical step in the appearance of epimerization activity has likely been the insertion of three residues near the C-terminus, which allow repositioning of the substrate in front of the iron center. The loss of the original carbocyclization activity has then occurred with changes in four amino acids near the iron center that prohibit alignment of the substrate for the formation of the C2-C5″ bond. Our study provides detailed insights into the evolutionary processes that have enabled Streptomyces soil bacteria to become the major source of antibiotics and antiproliferative agents. ENZYMES: EC number 1.14.11.

Nitrene Transfer Catalyzed by a Non-Heme Iron Enzyme and Enhanced by Non-Native Small-Molecule Ligands.

Transition-metal catalysis is a powerful tool for the construction of chemical bonds. Here we show that Pseudomonas savastanoi ethylene-forming enzyme, a non-heme iron enzyme, can catalyze olefin aziridination and nitrene C-H insertion, and that these activities can be improved by directed evolution. The non-heme iron center allows for facile modification of the primary coordination sphere by addition of metal-coordinating molecules, enabling control over enzyme activity and selectivity using small molecules.

Analysis of MEMO1 Binding Specificity for ErbB2 Using Fluorescence Polarization and Molecular Dynamics Simulations.

ErbB2 signaling pathways are linked to breast cancer formation, growth, and aggression; therefore, understanding the behavior of proteins associated with these pathways as well as regulatory factors that influence ErbB2 function is essential. MEMO1 is a redox active protein that is shown to associate with phosphorylated ErbB2 and mediate cell motility. We have developed a fluorescence polarization assay to probe the interaction between MEMO1 and an ErbB2-derived peptide containing a phosphorylated tyrosine residue. This interaction is shown to be pH-dependent and stronger with longer peptides as would be expected for protein-protein interactions. We have quantitatively mapped the binding interface of MEMO1 to the peptide using the fluorescence polarization assay and molecular dynamics simulations. We have confirmed that phosphorylation of the peptide is essential for binding and through mutagenesis have identified residues that contribute to favorable interactions. Our results highlight the importance of the protein-protein interactions of MEMO1 that complement the oxidase activity. In the future, these studies will provide a method for screening for selective modulators of MEMO1, which will allow for additional biological investigations.

HRG/HER2/HER3 signaling promotes AhR-mediated Memo-1 expression and migration in colorectal cancer.

Colorectal cancer (CRC) is a complex disease with still unsatisfactory prognosis even in western societies, although substantial progress has been made in pre-screening programs, surgical techniques and targeted therapy options. Mediator of motility-1 (Memo-1) was previously recognized as an important effector of cell migration downstream of receptor tyrosine kinase signaling in breast cancer. This study identified Memo-1 as frequently overexpressed in CRC and established a close link between extracellular HER2 activation, AhR/ARNT transcriptional activity and Memo-1 expression. Dissection of the hMemo-1 gene promoter using reporter assays and chromatin IP techniques revealed recruitment of Aryl hydrocarbon receptor (AhR)/Aryl hydrocarbon receptor nuclear-translocator (ARNT) complex, which positively influenced Memo-1 expression in cancer cells. We found that Memo-1 depletion negatively influenced the cellular actin network and that its expression is required for HER2-mediated cell migration and invasion. Moreover, analyses of Memo-1 expression in primary CRC revealed correlation with clinical parameters that point to Memo-1 as a new prognostic factor of aggressive disease in CRC patients. Altogether, these observations demonstrate that Memo-1 is an important downstream regulator of HER2-driven CRC cell migration and invasion through connecting extracellular signals from membrane to the cytoskeletal actin network.

Designing Covalently Linked Heterodimeric Four-Helix Bundles.

De novo design has proven a powerful methodology for understanding protein folding and function, and for mimicking or even bettering the properties of natural proteins. Extensive progress has been made in the design of helical bundles, simple structural motifs that can be nowadays designed with a high degree of precision. Among helical bundles, the four-helix bundle is widespread in nature, and is involved in numerous and fundamental processes. Representative examples are the carboxylate bridged diiron proteins, which perform a variety of different functions, ranging from reversible dioxygen binding to catalysis of dioxygen-dependent reactions, including epoxidation, desaturation, monohydroxylation, and radical formation. The "Due Ferri" (two-irons; DF) family of proteins is the result of a de novo design approach, aimed to reproduce in minimal four-helix bundle models the properties of the more complex natural diiron proteins, and to address how the amino acid sequence modulates their functions. The results so far obtained point out that asymmetric metal environments are essential to reprogram functions, and to achieve the specificity and selectivity of the natural enzymes. Here, we describe a design method that allows constructing asymmetric four-helix bundles through the covalent heterodimerization of two different α-helical harpins. In particular, starting from the homodimeric DF3 structure, we developed a protocol for covalently linking the two α2 monomers by using the Cu(I) catalyzed azide-alkyne cycloaddition. The protocol was then generalized, in order to include the construction of several linkers, in different protein positions. Our method is fast, low cost, and in principle can be applied to any couple of peptides/proteins we desire to link.

Memo interacts with c-Src to control Estrogen Receptor alpha sub-cellular localization.

Understanding the complex interaction between growth factor and steroid hormone signaling pathways in breast cancer is key to identifying suitable therapeutic strategies to avoid progression and therapy resistance. The interaction between these two pathways is of paramount importance for the development of endocrine resistance. Nevertheless, the molecular mechanisms behind their crosstalk are still largely obscure. We previously reported that Memo is a small redox-active protein that controls heregulin-mediated migration of breast cancer cells. Here we report that Memo sits at the intersection between heregulin and estrogen signaling, and that Memo controls Estrogen Receptor alpha (ERα) sub-cellular localization, phosphorylation, and function downstream of heregulin and estrogen in breast cancer cells. Memo facilitates ERα and c-Src interaction, ERα Y537 phosphorylation, and has the ability to control ERα extra-nuclear localization. Thus, we identify Memo as an important key mediator between the heregulin and estrogen signaling pathways, which affects both breast cancer cell migration and proliferation.

Effect of Outer-Sphere Side Chain Substitutions on the Fate of the trans Iron-Nitrosyl Dimer in Heme/Nonheme Engineered Myoglobins (Fe(B)Mbs): Insights into the Mechanism of Denitrifying NO Reductases.

Denitrifying NO reductases are transmembrane protein complexes that utilize a heme/nonheme diiron center at their active sites to reduce two NO molecules to the innocuous gas N2O. Fe(B)Mb proteins, with their nonheme iron sites engineered into the heme distal pocket of sperm whale myoglobin, are attractive models for studying the molecular details of the NO reduction reaction. Spectroscopic and structural studies of Fe(B)Mb constructs have confirmed that they reproduce the metal coordination spheres observed at the active site of the cytochrome c-dependent NO reductase from Pseudomonas aeruginosa. Exposure of Fe(B)Mb to excess NO, as examined by analytical and spectroscopic techniques, results primarily in the formation of a five-coordinate heme-nitrosyl complex without N2O production. However, substitution of the outer-sphere residue Ile107 with a glutamic acid (i.e., I107E) decreases the formation rate of the five-coordinate heme-nitrosyl complex and allows for the substoichiometric production of N2O. Here, we aim to better characterize the formation of the five-coordinate heme-nitrosyl complex and to explain why the level of N2O production increases with the I107E substitution. We follow the formation of the five-coordinate heme-nitrosyl inhibitory complex through the sequential exposure of Fe(B)Mb to different NO isotopomers using rapid-freeze-quench resonance Raman spectroscopy. The data show that the complex is formed by the displacement of the proximal histidine by a new NO molecule after the weakening of the Fe(II)-His bond in the intermediate six-coordinate low-spin (6cLS) heme-nitrosyl complex. These results lead us to explore diatomic migration within the scaffold of myoglobin and whether substitutions at residue 107 can be sufficient to control access to the proximal heme cavities. Results on a new Fe(B)Mb construct with an I107F substitution (Fe(B)Mb3) show an increased rate for the formation of the five-coordinate low-spin heme-nitrosyl complex without N2O production. Taken together, our results suggest that production of N2O from the [6cLS heme {FeNO}(7)/{Fe(B)NO}(7)] trans iron-nitrosyl dimer intermediate requires a proton transfer event facilitated by an outer-sphere residue such as E107 in Fe(B)Mb2 and E280 in P. aeruginosa cNOR.

Coq7p relevant residues for protein activity and stability.

Coenzyme Q (Q) is an isoprenylated benzoquinone electron carrier required for electronic transport in the mitochondrial respiratory chain, shuttling electrons from complexes I and II to complex III. Q synthesis requires proteins termed Coq (Coq1-Coq11). Coq7p is part of the multimeric complex involved in Q synthesis catalyzing the hydroxylation of demethoxy-Q6 (DMQ6), the last monooxygenase step in Q synthesis with a catalytic center containing a carboxylate-bridged di-iron at the active site of the enzyme. Here we indicate a group of Coq7p residues that modulate protein activity: D53, R57, V111 and S114. R57, V111 and S114 are very conserved residues; V111 and S114 are present in separated communities of amino acid correlation analysis. The coq7 double mutant V111G/S114A and S114E show respiratory deficiency at non permissive temperature, DMQ6 accumulation and lower content of Q6. Therefore we conclude that phosphomimetic S114E inhibit Coq7p activity, and propose that S114 phosphorylation is required to move a non-structured loop of 25 amino acids between helix 2 and 3, and that affects the di-iron coordination in Coq7p catalytic center.

[Prokaryotic expression, purification and activity detection of the extracellular and intracellular domains of human IGF1R beta subunit].

OBJECTIVE: To construct the prokaryotic expression vectors of extracellular domain (ß-ED) and intracellular protein kinase domain (ß-PKD) of insulin-like growth factor 1 receptor beta (IGF1R-ß) subunit, purify the fusion proteins GST-IGF1R ß-ED and GST-IGF1R ß-PKD, and detect their activities. METHODS: Human GST-IGF1R ß-ED and GST-IGF1R ß-PKD coding regions were amplified from human mammary cDNA library by PCR and cloned into the prokaryotic expression vector pGEX-KG. The fusion proteins GST-IGF1R ß-ED and GST-IGF1R ß-PKD were expressed in E.coli Rossate and purified by GST-Sepharose 4B beads. The expression of the fusion proteins were detected by Western blotting. The interactions of the proteins with mediator of epidermal growth factor receptor-2 (ERBB2)-driven cell motility (MEMO) protein were identified by GST pull-down assay. RESULTS: GST-IGF1R ß-ED and GST-IGF1R ß-PKD recombinant plasmids were successfully cloned. Double enzyme digestion and sequencing confirmed that the inserted fragments were identical to the target ones. The fusion proteins were successfully induced in Rossate and Western blotting showed the expression as expected. GST pull-down assay revealed that GST-IGF1R ß-PKD could interact with MEMO in vitro. CONCLUSION: GST-IGF1R ß-ED and GST-IGF1R ß-PKD were successfully cloned and purified. In addition, GST-IGF1R ß-PKD could interact with MEMO in vitro, which demonstrated the good activity of the purified proteins.

Iron homeostasis in host defence and inflammation.

Iron is an essential trace element for multicellular organisms and nearly all microorganisms. Although iron is abundant in the environment, common forms of iron are minimally soluble and therefore poorly accessible to biological organisms. Microorganisms entering a mammalian host face multiple mechanisms that further restrict their ability to obtain iron and thereby limit their pathogenicity. Iron levels also modulate host defence, as iron content in macrophages regulates their cytokine production. Here, we review recent advances that highlight the role of systemic and cellular iron-regulating mechanisms in protecting hosts from infection, emphasizing aspects that are applicable to human health and disease.

Occurrence of Isopenicillin-N-Synthase Homologs in Bioluminescent Ctenophores and Implications for Coelenterazine Biosynthesis.

The biosynthesis of the luciferin coelenterazine has remained a mystery for decades. While not all organisms that use coelenterazine appear to make it themselves, it is thought that ctenophores are a likely producer. Here we analyze the transcriptome data of 24 species of ctenophores, two of which have published genomes. The natural precursors of coelenterazine have been shown to be the amino acids L-tyrosine and L-phenylalanine, with the most likely biosynthetic pathway involving cyclization and further modification of the tripeptide Phe-Tyr-Tyr ("FYY"). Therefore, we searched the ctenophore transcriptome data for genes with the short peptide "FYY" as part of their coding sequence. We recovered a group of candidate genes for coelenterazine biosynthesis in the luminous species which encode a set of highly conserved non-heme iron oxidases similar to isopenicillin-N-synthase. These genes were absent in the transcriptomes and genome of the two non-luminous species. Pairwise identities and substitution rates reveal an unusually high degree of identity even between the most unrelated species. Additionally, two related groups of non-heme iron oxidases were found across all ctenophores, including those which are non-luminous, arguing against the involvement of these two gene groups in luminescence. Important residues for iron-binding are conserved across all proteins in the three groups, suggesting this function is still present. Given the known functions of other members of this protein superfamily are involved in heterocycle formation, we consider these genes to be top candidates for laboratory characterization or gene knockouts in the investigation of coelenterazine biosynthesis.