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Nature Communications Jan 2022Rieske oxygenases exploit the reactivity of iron to perform chemically challenging C-H bond functionalization reactions. Thus far, only a handful of Rieske oxygenases...
Rieske oxygenases exploit the reactivity of iron to perform chemically challenging C-H bond functionalization reactions. Thus far, only a handful of Rieske oxygenases have been structurally characterized and remarkably little information exists regarding how these enzymes use a common architecture and set of metallocenters to facilitate a diverse range of reactions. Herein, we detail how two Rieske oxygenases SxtT and GxtA use different protein regions to influence the site-selectivity of their catalyzed monohydroxylation reactions. We present high resolution crystal structures of SxtT and GxtA with the native β-saxitoxinol and saxitoxin substrates bound in addition to a Xenon-pressurized structure of GxtA that reveals the location of a substrate access tunnel to the active site. Ultimately, this structural information allowed for the identification of six residues distributed between three regions of SxtT that together control the selectivity of the C-H hydroxylation event. Substitution of these residues produces a SxtT variant that is fully adapted to exhibit the non-native site-selectivity and substrate scope of GxtA. Importantly, we also found that these selectivity regions are conserved in other structurally characterized Rieske oxygenases, providing a framework for predictively repurposing and manipulating Rieske oxygenases as biocatalysts.
Topics: Catalytic Domain; Crystallography, X-Ray; Escherichia coli; Escherichia coli Proteins; Hydroxylation; Iron; Models, Molecular; Oxygenases; Substrate Specificity; Trans-Activators
PubMed: 35017498
DOI: 10.1038/s41467-021-27822-3 -
Molecules (Basel, Switzerland) Apr 2020Abundant in nature, carotenoids are a class of fat-soluble pigments with a polyene tetraterpenoid structure. They possess antioxidant properties and their consumption...
Abundant in nature, carotenoids are a class of fat-soluble pigments with a polyene tetraterpenoid structure. They possess antioxidant properties and their consumption leads to certain health benefits in humans. Carotenoid cleavage oxygenases (CCOs) are a superfamily of enzymes which oxidatively cleave carotenoids and they are present in all kingdoms of life. Complexity of CCO evolution is high. For example, in this study we serendipitously found a new family of eukaryotic CCOs, the apocarotenoid oxygenase-like (ACOL) family. This family has several members in animal genomes and lacks the animal-specific amino acid motif PDPCK. This motif is likely to be associated with palmitoylation of some animal CCOs. We recently demonstrated that two mammalian members of the carotenoid oxygenase family retinal pigment epithelial-specific 65 kDa protein (RPE65) and beta-carotene oxygenase 2 (BCO2) are palmitoylated proteins. Here we used the acyl-resin-assisted capture (acyl-RAC) method to demonstrate protein palmitoylation and immunochemistry to localize mouse BCO2 (mBCO2) in COS7 cell line in the absence and presence of its substrate β-carotene. We demonstrate that mBCO2 palmitoylation depends on the evolutionarily conserved motif PDPCK and that metazoan family members lacking the motif (Lancelet beta-carotene oxygenase-like protein (BCOL) and Acropora ACOL) are not palmitoylated. Additionally, we observed that the palmitoylation status of mBCO2 and its membrane association depend on the presence of its substrate β-carotene. Based on our results we conclude that most metazoan carotenoid oxygenases retain the evolutionarily conserved palmitoylation PDPCK motif to target proteins to internal membranes depending on substrate status. Exceptions are in the secreted BCOL subfamily and the strictly cytosolic ancient ACOL subfamily of carotenoid oxygenases.
Topics: Animals; Carotenoids; Dioxygenases; Fatty Acids, Monounsaturated; Fluorescent Antibody Technique; Humans; Mice; Multigene Family; Mutation; Oxygenases; Phylogeny; Protein Transport; Substrate Specificity
PubMed: 32331396
DOI: 10.3390/molecules25081942 -
Ruffling is essential for Staphylococcus aureus IsdG-catalyzed degradation of heme to staphylobilin.Journal of Inorganic Biochemistry May 2022Non-canonical heme oxygenases are enzymes that degrade heme to non-biliverdin products within bacterial heme iron acquisition pathways. These enzymes all contain a...
Non-canonical heme oxygenases are enzymes that degrade heme to non-biliverdin products within bacterial heme iron acquisition pathways. These enzymes all contain a conserved second-sphere Trp residue that is essential for enzymatic turnover. Here, UV/Vis absorption (Abs) and circular dichroism (CD) spectroscopies were employed to show that the W67F variant of IsdG perturbs the heme substrate conformation. In general, a dynamic equilibrium between "planar" and "ruffled" substrate conformations exists within non-canonical heme oxygenases, and that the second-sphere Trp favors population of the "ruffled" substrate conformation. H nuclear magnetic resonance and magnetic CD spectroscopies were used to characterize the electronic structures of IsdG and IsdI variants with different substrate conformational distributions. These data revealed that the "ruffled" substrate conformation promotes partial porphyrin-to‑iron electron transfer, which makes the meso carbons of the porphyrin ring susceptible to radical attack. Finally, UV/Vis Abs spectroscopy was utilized to quantify the enzymatic rates, and electrospray ionization mass spectrometry was used to identify the product distributions, for variants of IsdG with altered substrate conformational distributions. In general, the rate of heme oxygenation by non-canonical heme oxygenases depends upon the population of the "ruffled" substrate conformation. Also, the production of staphylobilin or mycobilin by these enzymes is correlated with the population of the "ruffled" substrate conformation, since variants that favor population of the "planar" substrate conformation yield significant amounts of biliverdin. These data can be understood within the framework of a concerted rearrangement mechanism for the monooxygenation of heme to meso-hydroxyheme by non-canonical heme oxygenases.
Topics: Bacterial Proteins; Catalysis; Heme; Heme Oxygenase (Decyclizing); Iron; Oxygenases; Staphylococcus aureus
PubMed: 35247855
DOI: 10.1016/j.jinorgbio.2022.111775 -
Natural Product Reports Jul 2018Covering: up to 2017 The participation of non-heme dinuclear iron cluster-containing monooxygenases in natural product biosynthetic pathways has been recognized only... (Review)
Review
Covering: up to 2017 The participation of non-heme dinuclear iron cluster-containing monooxygenases in natural product biosynthetic pathways has been recognized only recently. At present, two families have been discovered. The archetypal member of the first family, CmlA, catalyzes β-hydroxylation of l-p-aminophenylalanine (l-PAPA) covalently linked to the nonribosomal peptide synthetase (NRPS) CmlP, thereby effecting the first step in the biosynthesis of chloramphenicol by Streptomyces venezuelae. CmlA houses the diiron cluster in a metallo-β-lactamase protein fold instead of the 4-helix bundle fold of nearly every other diiron monooxygenase. CmlA couples O2 activation and substrate hydroxylation via a structural change caused by formation of the l-PAPA-loaded CmlP:CmlA complex. The other new diiron family is typified by two enzymes, AurF and CmlI, which catalyze conversion of aryl-amine substrates to aryl-nitro products with incorporation of oxygen from O2. AurF from Streptomyces thioluteus catalyzes the formation of p-nitrobenzoate from p-aminobenzoate as a precursor to the biostatic compound aureothin, whereas CmlI from S. venezuelae catalyzes the ultimate aryl-amine to aryl-nitro step in chloramphenicol biosynthesis. Both enzymes stabilize a novel type of peroxo-intermediate as the reactive species. The rare 6-electron N-oxygenation reactions of CmlI and AurF involve two progressively oxidized pathway intermediates. The enzymes optimize efficiency by utilizing one of the reaction pathway intermediates as an in situ reductant for the diiron cluster, while simultaneously generating the next pathway intermediate. For CmlI, this reduction allows mid-pathway regeneration of the peroxo intermediate required to complete the biosynthesis. CmlI ensures specificity by carrying out the multistep aryl-amine oxygenation without dissociating intermediate products.
Topics: Bacterial Proteins; Biological Products; Biosynthetic Pathways; Chloramphenicol; Crystallography, X-Ray; Kinetics; Mixed Function Oxygenases; Oxygen; Oxygenases; Peptide Synthases; Protein Conformation
PubMed: 29552683
DOI: 10.1039/C7NP00061H -
FEMS Microbiology Reviews Oct 2003Based on structural, biochemical, and genetic data, the soluble diiron monooxygenases can be divided into four groups: the soluble methane monooxygenases, the Amo alkene... (Review)
Review
Based on structural, biochemical, and genetic data, the soluble diiron monooxygenases can be divided into four groups: the soluble methane monooxygenases, the Amo alkene monooxygenase of Rhodococcus corallinus B-276, the phenol hydroxylases, and the four-component alkene/aromatic monooxygenases. The limited phylogenetic distribution of these enzymes among bacteria, together with available genetic evidence, indicates that they have been spread largely through horizontal gene transfer. Phylogenetic analyses reveal that the alpha- and beta-oxygenase subunits are paralogous proteins and were derived from an ancient gene duplication of a carboxylate-bridged diiron protein, with subsequent divergence yielding a catalytic alpha-oxygenase subunit and a structural beta-oxygenase subunit. The oxidoreductase and ferredoxin components of these enzymes are likely to have been acquired by horizontal transfer from ancestors common to unrelated diiron and Rieske center oxygenases and other enzymes. The cumulative results of phylogenetic reconstructions suggest that the alkene/aromatic monooxygenases diverged first from the last common ancestor for these enzymes, followed by the phenol hydroxylases, Amo alkene monooxygenase, and methane monooxygenases.
Topics: Amino Acid Sequence; Evolution, Molecular; Iron; Mixed Function Oxygenases; Molecular Sequence Data; Oxygenases; Rhodococcus; Solubility
PubMed: 14550940
DOI: 10.1016/S0168-6445(03)00023-8 -
Archives of Biochemistry and Biophysics Nov 2013Carotenoids and their metabolic derivatives serve critical functions in both prokaryotic and eukaryotic cells, including pigmentation, photoprotection and photosynthesis... (Review)
Review
Carotenoids and their metabolic derivatives serve critical functions in both prokaryotic and eukaryotic cells, including pigmentation, photoprotection and photosynthesis as well as cell signaling. These organic compounds are also important for visual function in vertebrate and non-vertebrate organisms. Enzymatic transformations of carotenoids to various apocarotenoid products are catalyzed by a family of evolutionarily conserved, non-heme iron-containing enzymes named carotenoid cleavage oxygenases (CCOs). Studies have revealed that CCOs are critically involved in carotenoid homeostasis and essential for the health of organisms including humans. These enzymes typically display a high degree of regio- and stereo-selectivity, acting on specific positions of the polyene backbone located in their substrates. By oxidatively cleaving and/or isomerizing specific double bonds, CCOs generate a variety of apocarotenoid isomer products. Recent structural studies have helped illuminate the mechanisms by which CCOs mobilize their lipophilic substrates from biological membranes to perform their characteristic double bond cleavage and/or isomerization reactions. In this review, we aim to integrate structural and biochemical information about CCOs to provide insights into their catalytic mechanisms.
Topics: Animals; Bacteria; Bacterial Physiological Phenomena; Carotenoids; Catalysis; Humans; Mammals; Oxygenases; Stereoisomerism; Substrate Specificity
PubMed: 23827316
DOI: 10.1016/j.abb.2013.06.012 -
Microbial Cell Factories Dec 2022The use of biologically produced alkanes has attracted considerable attention as an alternative energy source to petroleum. In 2010, the alkane synthesis pathway in... (Review)
Review
The use of biologically produced alkanes has attracted considerable attention as an alternative energy source to petroleum. In 2010, the alkane synthesis pathway in cyanobacteria was found to include two small globular proteins, acyl-(acyl carrier protein [ACP]) reductase (AAR) and aldehyde deformylating oxygenase (ADO). AAR produces fatty aldehydes from acyl-ACPs/CoAs, which are then converted by ADO to alkanes/alkenes equivalent to diesel oil. This discovery has paved the way for alkane production by genetically modified organisms. Since then, many studies have investigated the reactions catalyzed by AAR and ADO. In this review, we first summarize recent findings on structures and catalytic mechanisms of AAR and ADO. We then outline the mechanism by which AAR and ADO form a complex and efficiently transfer the insoluble aldehyde produced by AAR to ADO. Furthermore, we describe recent advances in protein engineering studies on AAR and ADO to improve the efficiency of alkane production in genetically engineered microorganisms such as Escherichia coli and cyanobacteria. Finally, the role of alkanes in cyanobacteria and future perspectives for bioalkane production using AAR and ADO are discussed. This review provides strategies for improving the production of bioalkanes using AAR and ADO in cyanobacteria for enabling the production of carbon-neutral fuels.
Topics: Cyanobacteria; Oxygenases; Alkanes; Oxidoreductases; Escherichia coli; Aldehydes
PubMed: 36503511
DOI: 10.1186/s12934-022-01981-4 -
Cell Metabolism Jan 2009Aerobic organisms developed mechanisms to protect themselves against a shortage of oxygen (O(2)). Recent studies reveal that O(2) sensors, belonging to the novel class... (Review)
Review
Aerobic organisms developed mechanisms to protect themselves against a shortage of oxygen (O(2)). Recent studies reveal that O(2) sensors, belonging to the novel class of 2-oxoglutarate dependent iron(ii)-dioxygenases, have more important roles in metabolism than anticipated. Here, we provide a "metabolo-centric" overview of the role of the PHD/FIH members of this family in metabolism, in particular on how they regulate O(2) supply and consumption, energy compensation and conservation, O(2) conformance and hypoxia tolerance, redox and pH homeostasis, and other vital metabolic processes with implications in health and disease. These insights may offer novel opportunities for the treatment of ischemic diseases.
Topics: Animals; Fatty Acids; Glucose; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemia; Mice; Oxygen Consumption; Oxygenases; Procollagen-Proline Dioxygenase; Signal Transduction
PubMed: 19117543
DOI: 10.1016/j.cmet.2008.10.001 -
Biochimica Et Biophysica Acta Aug 2011Chlorophyll breakdown is an important catabolic process of leaf senescence and fruit ripening. Structure elucidation of colorless linear tetrapyrroles as (final)... (Review)
Review
Chlorophyll breakdown is an important catabolic process of leaf senescence and fruit ripening. Structure elucidation of colorless linear tetrapyrroles as (final) breakdown products of chlorophyll was crucial for the recent delineation of a chlorophyll breakdown pathway which is highly conserved in land plants. Pheophorbide a oxygenase is the key enzyme responsible for opening of the chlorin macrocycle of pheophorbide a characteristic to all further breakdown products. Degradation of chlorophyll was rationalized by the need of a senescing cell to detoxify the potentially phototoxic pigment, yet recent investigations in leaves and fruits indicate that chlorophyll catabolites could have physiological roles. This review updates structural information of chlorophyll catabolites and the biochemical reactions involved in their formation, and discusses the significance of chlorophyll breakdown. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.
Topics: Chlorophyll; Chlorophyll A; Oxygenases; Plants
PubMed: 21167811
DOI: 10.1016/j.bbabio.2010.12.007 -
Journal of Biological Inorganic... Apr 2017A wide range of spectroscopic approaches have been used to interrogate the mononuclear iron metallocenter in 2-oxoglutarate (2OG)-dependent oxygenases. The results from... (Review)
Review
A wide range of spectroscopic approaches have been used to interrogate the mononuclear iron metallocenter in 2-oxoglutarate (2OG)-dependent oxygenases. The results from these spectroscopic studies have provided valuable insights into the structural changes at the active site during substrate binding and catalysis, thus providing critical information that complements investigations of these enzymes by X-ray crystallography, biochemical, and computational approaches. This mini-review highlights taurine hydroxylase (taurine:2OG dioxygenase, TauD) as a case study to illustrate the wealth of knowledge that can be generated by applying a diverse array of spectroscopic investigations to a single enzyme. In particular, electronic absorption, circular dichroism, magnetic circular dichroism, conventional and pulse electron paramagnetic, Mössbauer, X-ray absorption, and resonance Raman methods have been exploited to uncover the properties of the metal site in TauD.
Topics: Mixed Function Oxygenases; Spectrum Analysis
PubMed: 27812832
DOI: 10.1007/s00775-016-1406-3