Nature’s Machinery, Repurposed: Expanding the Repertoire of Iron-Dependent Oxygenases

Iron is an especially important redox-active cofactor in biology because of its ability to mediate reactions with atmospheric O₂. Iron-dependent oxygenases exploit this earth-abundant transition metal for the insertion of oxygen atoms into organic compounds. Throughout the astounding diversity of transformations catalyzed by these enzymes, the protein framework directs reactive intermediates toward the precise formation of products, which, in many cases, necessitates the cleavage of strong C–H bonds. In recent years, members of several iron-dependent oxygenase families have been engineered for new-to-nature transformations that offer advantages over conventional synthetic methods. In this Perspective, we first explore what is known about the reactivity of heme-dependent cytochrome P450 oxygenases and nonheme iron-dependent oxygenases bearing the 2-His-1-carboxylate facial triad by reviewing mechanistic studies with an emphasis on how the protein scaffold maximizes the catalytic potential of the iron-heme and iron cofactors. We then review how these cofactors have been repurposed for abiological transformations by engineering the protein frameworks of these enzymes. Finally, we discuss contemporary challenges associated with engineering these platforms and comment on their roles in biocatalysis moving forward

Nature’s Machinery, Repurposed: Expanding the Repertoire of Iron-Dependent Oxygenases

Iron is an especially important redox-active cofactor in biology because of its ability to mediate reactions with atmospheric O₂. Iron-dependent oxygenases exploit this earth-abundant transition metal for the insertion of oxygen atoms into organic compounds. Throughout the astounding diversity of transformations catalyzed by these enzymes, the protein framework directs reactive intermediates toward the precise formation of products, which, in many cases, necessitates the cleavage of strong C–H bonds. In recent years, members of several iron-dependent oxygenase families have been engineered for new-to-nature transformations that offer advantages over conventional synthetic methods. In this Perspective, we first explore what is known about the reactivity of heme-dependent cytochrome P450 oxygenases and nonheme iron-dependent oxygenases bearing the 2-His-1-carboxylate facial triad by reviewing mechanistic studies with an emphasis on how the protein scaffold maximizes the catalytic potential of the iron-heme and iron cofactors. We then review how these cofactors have been repurposed for abiological transformations by engineering the protein frameworks of these enzymes. Finally, we discuss contemporary challenges associated with engineering these platforms and comment on their roles in biocatalysis moving forward

Hydrogen Donation but not Abstraction by a Tyrosine (Y68) During Endoperoxide Installation by Verruculogen Synthase (FtmOx1)

Hydrogen-atom transfer (HAT) from a substrate carbon to an iron(IV)-oxo (ferryl) intermediate initiates a diverse array of enzymatic transformations. For outcomes other than hydroxylation, coupling of the resultant carbon radical and hydroxo ligand (oxygen rebound) must generally be averted. A recent study of FtmOx1, a fungal iron(II)- and 2-(oxo)glutarate-dependent oxygenase that installs the endoperoxide of verruculogen by adding O_2 between carbons 21 and 27 of fumitremorgin B, posited that tyrosine (Tyr or Y) 224 serves as HAT intermediary to separate the C21 radical (C21•) and Fe(III)–OH HAT products and prevent rebound. Our reinvestigation of the FtmOx1 mechanism revealed, instead, direct HAT from C21 to the ferryl complex and surprisingly competitive rebound. The C21-hydroxylated (rebound) product, which undergoes deprenylation, predominates when low [O_2] slows C21•–O_2 coupling in the next step of the endoperoxidation pathway. This pathway culminates with addition of the C21–O–O• peroxyl adduct to olefinic C27 followed by HAT to the C26• from a Tyr. The last step results in sequential accumulation of Tyr radicals, which are suppressed without detriment to turnover by inclusion of the reductant, ascorbate. Replacement of each of four candidates for the proximal C26 H• donor (including Y224) with phenylalanine (F) revealed that only the Y68F variant (i) fails to accumulate the first Tyr• and (ii) makes an altered major product, identifying Y68 as the donor. The implied proximities of C21 to the iron cofactor and C26 to Y68 support a new docking model of the enzyme–substrate complex that is consistent with all available data

A many-analysts approach to the relation between religiosity and well-being

The relation between religiosity and well-being is one of the most researched topics in the psychology of religion, yet the directionality and robustness of the effect remains debated. Here, we adopted a many-analysts approach to assess the robustness of this relation based on a new cross-cultural dataset (N=10,535 participants from 24 countries). We recruited 120 analysis teams to investigate (1) whether religious people self-report higher well-being, and (2) whether the relation between religiosity and self-reported well-being depends on perceived cultural norms of religion (i.e., whether it is considered normal and desirable to be religious in a given country). In a two-stage procedure, the teams first created an analysis plan and then executed their planned analysis on the data. For the first research question, all but 3 teams reported positive effect sizes with credible/confidence intervals excluding zero (median reported β=0.120). For the second research question, this was the case for 65% of the teams (median reported β=0.039). While most teams applied (multilevel) linear regression models, there was considerable variability in the choice of items used to construct the independent variables, the dependent variable, and the included covariates

A Many-analysts Approach to the Relation Between Religiosity and Well-being

The relation between religiosity and well-being is one of the most researched topics in the psychology of religion, yet the directionality and robustness of the effect remains debated. Here, we adopted a many-analysts approach to assess the robustness of this relation based on a new cross-cultural dataset (N = 10, 535 participants from 24 countries). We recruited 120 analysis teams to investigate (1) whether religious people self-report higher well-being, and (2) whether the relation between religiosity and self-reported well-being depends on perceived cultural norms of religion (i.e., whether it is considered normal and desirable to be religious in a given country). In a two-stage procedure, the teams first created an analysis plan and then executed their planned analysis on the data. For the first research question, all but 3 teams reported positive effect sizes with credible/confidence intervals excluding zero (median reported β = 0.120). For the second research question, this was the case for 65% of the teams (median reported β = 0.039). While most teams applied (multilevel) linear regression models, there was considerable variability in the choice of items used to construct the independent variables, the dependent variable, and the included covariates

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The Cation−π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification

The design of fluorogenic probes for a Halo tag is highly desirable but challenging. Previous work achieved this goal by controlling the chemical switch of spirolactones upon the covalent conjugation between the Halo tag and probes or by incorporating a “channel dye” into the substrate binding tunnel of the Halo tag. In this work, we have developed a novel class of Halo-tag fluorogenic probes that are derived from solvatochromic fluorophores. The optimal probe, harboring a benzothiadiazole scaffold, exhibits a 1000-fold fluorescence enhancement upon reaction with the Halo tag. Structural, computational, and biochemical studies reveal that the benzene ring of a tryptophan residue engages in a cation−π interaction with the dimethylamino electron-donating group of the benzothiadiazole fluorophore in its excited state. We further demonstrate using noncanonical fluorinated tryptophan that the cation−π interaction directly contributes to the fluorogenicity of the benzothiadiazole fluorophore. Mechanistically, this interaction could contribute to the fluorogenicity by promoting the excited-state charge separation and inhibiting the twisting motion of the dimethylamino group, both leading to an enhanced fluorogenicity. Finally, we demonstrate the utility of the probe in no-wash direct imaging of Halo-tagged proteins in live cells. In addition, the fluorogenic nature of the probe enables a gel-free quantification of fusion proteins expressed in mammalian cells, an application that was not possible with previously nonfluorogenic Halo-tag probes. The unique mechanism revealed by this work suggests that incorporation of an excited-state cation−π interaction could be a feasible strategy for enhancing the optical performance of fluorophores and fluorogenic sensors

Structure-Guided Reprogramming of a Hydroxylase To Halogenate Its Small Molecule Substrate

Enzymatic installation of chlorine/bromine into unactivated carbon centers provides a versatile, selective, and environmentally friendly alternative to chemical halogenation. Iron­(II) and 2-(oxo)-glutarate (Fe^II/2OG)-dependent halogenases are powerful biocatalysts that are capable of cleaving aliphatic C–H bonds to introduce useful functional groups, including halogens. Using the structure of the Fe/2OG halogenase, WelO5, in complex with its small molecule substrate, we identified a similar <i>N</i>-acyl amino acid hydroxylase, SadA, and reprogrammed it to halogenate its substrate, thereby generating a new chiral haloalkyl center. The work highlights the potential of Fe^II/2OG enzymes as platforms for development of novel stereospecific catalysts for late-stage C–H functionalization

Two Distinct Mechanisms for C–C Desaturation by Iron(II)- and 2‑(Oxo)glutarate-Dependent Oxygenases: Importance of α‑Heteroatom Assistance

Hydroxylation of aliphatic carbons by nonheme Fe­(IV)-oxo (ferryl) complexes proceeds by hydrogen-atom (H•) transfer (HAT) to the ferryl and subsequent coupling between the carbon radical and Fe­(III)-coordinated oxygen (termed rebound). Enzymes that use H•-abstracting ferryl complexes for other transformations must either suppress rebound or further process hydroxylated intermediates. For olefin-installing C–C desaturations, it has been proposed that a second HAT to the Fe­(III)–OH complex from the carbon α to the radical preempts rebound. Deuterium (²H) at the second site should slow this step, potentially making rebound competitive. Desaturations mediated by two related l-arginine-modifying iron­(II)- and 2-(oxo)­glutarate-dependent (Fe/2OG) oxygenases behave oppositely in this key test, implicating different mechanisms. NapI, the l-Arg 4,5-desaturase from the naphthyridinomycin biosynthetic pathway, abstracts H• first from C5 but hydroxylates this site (leading to guanidine release) to the same modest extent whether C4 harbors ¹H or ²H. By contrast, an unexpected 3,4-desaturation of l-homoarginine (l-hArg) by VioC, the l-Arg 3-hydroxylase from the viomycin biosynthetic pathway, is markedly disfavored relative to C4 hydroxylation when C3 (the second hydrogen donor) harbors ²H. Anchimeric assistance by N6 permits removal of the C4–H as a proton in the NapI reaction, but, with no such assistance possible in the VioC desaturation, a second HAT step (from C3) is required. The close proximity (≤3.5 Å) of both l-hArg carbons to the oxygen ligand in an X-ray crystal structure of VioC harboring a vanadium-based ferryl mimic supports and rationalizes the sequential-HAT mechanism. The results suggest that, although the sequential-HAT mechanism is feasible, its geometric requirements may make competing hydroxylation unavoidable, thus explaining the presence of α-heteroatoms in nearly all native substrates for Fe/2OG desaturases