RESUMO
S-adenosylmethionine (AdoMet) has been referred to as both "a poor man's adenosylcobalamin (AdoCbl)" and "a rich man's AdoCbl," but today, with the ever-increasing number of functions attributed to each cofactor, both appear equally rich and surprising. The recent characterization of an organometallic species in an AdoMet radical enzyme suggests that the line that differentiates them in nature will be constantly challenged. Here, we compare and contrast AdoMet and cobalamin (Cbl) and consider why Cbl-dependent AdoMet radical enzymes require two cofactors that are so similar in their reactivity. We further carry out structural comparisons employing the recently determined crystal structure of oxetanocin-A biosynthetic enzyme OxsB, the first three-dimensional structural data on a Cbl-dependent AdoMet radical enzyme. We find that the structural motifs responsible for housing the AdoMet radical machinery are largely conserved, whereas the motifs responsible for binding additional cofactors are much more varied.
Assuntos
S-Adenosilmetionina/metabolismo , Vitamina B 12/metabolismo , Animais , Sítios de Ligação , Coenzimas/química , Coenzimas/metabolismo , Eletroquímica , Enzimas/química , Enzimas/metabolismo , Radicais Livres/química , Radicais Livres/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , S-Adenosilmetionina/química , Vitamina B 12/análogos & derivados , Vitamina B 12/químicaRESUMO
The 2011 discovery of the first rare earth-dependent enzyme in methylotrophic Methylobacterium extorquens AM1 prompted intensive research toward understanding the unique chemistry at play in these systems. This enzyme, an alcohol dehydrogenase (ADH), features a La3+ ion closely associated with redox-active coenzyme pyrroloquinoline quinone (PQQ) and is structurally homologous to the Ca2+-dependent ADH from the same organism. AM1 also produces a periplasmic PQQ-binding protein, PqqT, which we have now structurally characterized to 1.46-Å resolution by X-ray diffraction. This crystal structure reveals a Lys residue hydrogen-bonded to PQQ at the site analogously occupied by a Lewis acidic cation in ADH. Accordingly, we prepared K142A- and K142D-PqqT variants to assess the relevance of this site toward metal binding. Isothermal titration calorimetry experiments and titrations monitored by UV-Vis absorption and emission spectroscopies support that K142D-PqqT binds tightly (Kd = 0.6 ± 0.2 µM) to La3+ in the presence of bound PQQ and produces spectral signatures consistent with those of ADH enzymes. These spectral signatures are not observed for WT- or K142A-variants or upon addition of Ca2+ to PQQ ⸦ K142D-PqqT. Addition of benzyl alcohol to La3+-bound PQQ ⸦ K142D-PqqT (but not Ca2+-bound PQQ ⸦ K142D-PqqT, or La3+-bound PQQ ⸦ WT-PqqT) produces spectroscopic changes associated with PQQ reduction, and chemical trapping experiments reveal the production of benzaldehyde, supporting ADH activity. By creating a metal binding site that mimics native ADH enzymes, we present a rare earth-dependent artificial metalloenzyme primed for future mechanistic, biocatalytic, and biosensing applications.
Assuntos
Methylobacterium extorquens , Methylobacterium extorquens/enzimologia , Methylobacterium extorquens/metabolismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/química , Cristalografia por Raios X , Cofator PQQ/metabolismo , Cofator PQQ/química , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Metais Terras Raras/química , Metais Terras Raras/metabolismo , Modelos Moleculares , Lantânio/química , Lantânio/metabolismoRESUMO
Hydrogen-bonding (H-bonding) interactions in metalloprotein active sites can critically regulate enzyme function. Changes in the protein structure triggered by interplay with substrates, products, and partner proteins are often translated to the metallocofactor by way of specific changes in H-bond networks connected to the active site. However, the complexities of metalloprotein architecture and mechanism often preclude our ability to define the precise molecular interactions giving rise to these intricate regulatory pathways. To address this shortcoming, we have developed conformationally switchable artificial metalloproteins (swArMs) in which allosteric Gln-binding triggers protein conformational changes that impact the microenvironment surrounding an installed metallocofactor. Herein, we report a combined structural, spectroscopic, and computational approach to enhance the conformation-dependent changes in H-bond interactions surrounding the metallocofactor site of a swArM. Structure-informed molecular dynamics simulations were employed to predict point mutations that could enhance active site H-bond interactions preferentially in the Gln-bound holo-conformation of the swArM. Testing our predictions via the unique infrared spectral signals associated with the metallocofactor site, we have identified three key residues capable of imparting conformational control over the metallocofactor microenvironment. The resultant swArMs not only model biologically relevant structural regulation but also provide an enhanced Gln-responsive biological probe to be leveraged in future biosensing applications.
Assuntos
Ligação de Hidrogênio , Metaloproteínas , Simulação de Dinâmica Molecular , Conformação Proteica , Metaloproteínas/química , Metaloproteínas/metabolismo , Metaloproteínas/genética , Domínio CatalíticoRESUMO
Chlorophyll pigments are used by photosynthetic organisms to facilitate light capture and mediate the conversion of sunlight into chemical energy. Due to the indispensable nature of this pigment and its propensity to form reactive oxygen species, organisms heavily invest in its biosynthesis, recycling, and degradation. One key enzyme implicated in these processes is chlorophyllase, an α/ß hydrolase that hydrolyzes the phytol tail of chlorophyll pigments to produce chlorophyllide molecules. This enzyme was discovered a century ago, but despite its importance to diverse photosynthetic organisms, there are still many missing biochemical details regarding how chlorophyllase functions. Here, we present the 4.46-Å resolution crystal structure of chlorophyllase from Triticum aestivum. This structure reveals the dimeric architecture of chlorophyllase, the arrangement of catalytic residues, an unexpected divalent metal ion-binding site, and a substrate-binding site that can accommodate a diverse range of pigments. Further, this structure exhibits the existence of both intermolecular and intramolecular disulfide bonds. We investigated the importance of these architectural features using enzyme kinetics, mass spectrometry, and thermal shift assays. Through this work, we demonstrated that the oxidation state of the Cys residues is imperative to the activity and stability of chlorophyllase, illuminating a biochemical trigger for responding to environmental stress. Additional bioinformatics analysis of the chlorophyllase enzyme family reveals widespread conservation of key catalytic residues and the identified "redox switch" among other plant chlorophyllase homologs, thus revealing key details regarding the structure-function relationships in chlorophyllase.
Assuntos
Hidrolases de Éster Carboxílico , Clorofila , Triticum , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Clorofila/metabolismo , Dissulfetos , Triticum/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismoRESUMO
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.
Assuntos
Proteínas de Bactérias , Comamonas testosteroni , Oxigenases , Aldeído Desidrogenase/metabolismo , NAD/metabolismo , Oxigenases/metabolismo , Especificidade por Substrato , Comamonas testosteroni/enzimologia , Comamonas testosteroni/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ferroproteínas não Heme/química , Ferroproteínas não Heme/genética , Ferroproteínas não Heme/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estrutura Terciária de Proteína , Modelos Moleculares , Estabilidade Proteica , Biologia ComputacionalRESUMO
Rieske nonheme iron oxygenases use two metallocenters, a Rieske-type [2Fe-2S] cluster and a mononuclear iron center, to catalyze oxidation reactions on a broad range of substrates. These enzymes are widely used by microorganisms to degrade environmental pollutants and to build complexity in a myriad of biosynthetic pathways that are industrially interesting. However, despite the value of this chemistry, there is a dearth of understanding regarding the structure-function relationships in this enzyme class, which limits our ability to rationally redesign, optimize, and ultimately exploit the chemistry of these enzymes. Therefore, in this work, by leveraging a combination of available structural information and state-of-the-art protein modeling tools, we show that three "hotspot" regions can be targeted to alter the site selectivity, substrate preference, and substrate scope of the Rieske oxygenase p-toluenesulfonate methyl monooxygenase (TsaM). Through mutation of six to 10 residues distributed between three protein regions, TsaM was engineered to behave as either vanillate monooxygenase (VanA) or dicamba monooxygenase (DdmC). This engineering feat means that TsaM was rationally engineered to catalyze an oxidation reaction at the meta and ortho positions of an aromatic substrate, rather than its favored native para position, and that TsaM was redesigned to perform chemistry on dicamba, a substrate that is not natively accepted by the enzyme. This work thus contributes to unlocking our understanding of structure-function relationships in the Rieske oxygenase enzyme class and expands foundational principles for future engineering of these metalloenzymes.
Assuntos
Oxigenases de Função Mista , Oxigenases , Oxigenases/química , Oxigenases de Função Mista/metabolismo , Dicamba/metabolismo , Oxirredução , FerroRESUMO
Oxetanocin A (OXT-A) is a potent antitumour, antiviral and antibacterial compound. Biosynthesis of OXT-A has been linked to a plasmid-borne Bacillus megaterium gene cluster that contains four genes: oxsA, oxsB, oxrA and oxrB. Here we show that both the oxsA and oxsB genes are required for the production of OXT-A. Biochemical analysis of the encoded proteins, a cobalamin (Cbl)-dependent S-adenosylmethionine (AdoMet) radical enzyme, OxsB, and an HD-domain phosphohydrolase, OxsA, reveals that OXT-A is derived from a 2'-deoxyadenosine phosphate in an OxsB-catalysed ring contraction reaction initiated by hydrogen atom abstraction from C2'. Hence, OxsB represents the first biochemically characterized non-methylating Cbl-dependent AdoMet radical enzyme. X-ray analysis of OxsB reveals the fold of a Cbl-dependent AdoMet radical enzyme, a family of enzymes with an estimated 7,000 members. Overall, this work provides a framework for understanding the interplay of AdoMet and Cbl cofactors and expands the catalytic repertoire of Cbl-dependent AdoMet radical enzymes.
Assuntos
Adenina/análogos & derivados , Bacillus megaterium/enzimologia , Proteínas de Bactérias/metabolismo , Biocatálise , Coenzimas/metabolismo , S-Adenosilmetionina/metabolismo , Vitamina B 12/metabolismo , Adenina/biossíntese , Monofosfato de Adenosina/metabolismo , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Nucleotídeos de Desoxiadenina/metabolismo , Genes Bacterianos/genética , Modelos Moleculares , Família Multigênica/genética , Conformação ProteicaRESUMO
Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)2(X) cofactor (dmgH = dimethylglyoxime and X = N3-, H3C-, and iPr-). We used UV-vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co-S cysteine ligation within the Escherichia coli glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence, circular dichroism, and infrared spectroscopies, along with isothermal titration calorimetry, reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)2(CH3) cofactor, we show that the protein stabilizes the otherwise labile Co-S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.
Assuntos
Metaloproteínas , Metaloproteínas/química , Cristalografia por Raios X , Escherichia coli/metabolismo , Dicroísmo Circular , CinéticaRESUMO
The metabolic serine hydrolase family is, arguably, one of the largest functional enzyme classes in mammals, including humans, comprising 1-2% of the total proteome. This enzyme family uses a conserved nucleophilic serine residue in the active site to perform diverse hydrolytic reactions and consists of proteases, lipases, esterases, amidases, and transacylases, which are prototypical members of this family. In humans, this enzyme family consists of >250, of which approximately 40% members remain unannotated, in terms of both their endogenous substrates and the biological pathways that they regulate. The enzyme ABHD14B, an outlying member of this family, is also known as CCG1/TAFII250-interacting factor B, as it was found to be associated with transcription initiation factor TFIID. The crystal structure of human ABHD14B was determined more than a decade ago; however, its endogenous substrates remain elusive. In this paper, we annotate ABHD14B as a lysine deacetylase (KDAC), showing this enzyme's ability to transfer an acetyl group from a post-translationally acetylated lysine to coenzyme A (CoA), to yield acetyl-CoA, while regenerating the free amine of protein lysine residues. We validate these findings by in vitro biochemical assays using recombinantly purified human ABHD14B in conjunction with cellular studies in a mammalian cell line by knocking down ABHD14B and by identification of a putative substrate binding site. Finally, we report the development and characterization of a much-needed, exquisitely selective ABHD14B antibody, and using it, we map the cellular and tissue distribution of ABHD14B and prospective metabolic pathways that this enzyme might biologically regulate.
Assuntos
Acetiltransferases/metabolismo , Histona Acetiltransferases/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Acetilação , Acetiltransferases/química , Acetiltransferases/genética , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Coenzima A/química , Ensaios Enzimáticos , Escherichia coli/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Humanos , Hidrolases , Camundongos Endogâmicos C57BL , Coelhos , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/genéticaRESUMO
In humans, mitochondrial iron-sulfur cluster biosynthesis is an essential biochemical process mediated by the assembly complex consisting of cysteine desulfurase (NFS1), LYR protein (ISD11), acyl-carrier protein (ACP), and the iron-sulfur cluster assembly scaffold protein (ISCU2). The protein frataxin (FXN) is an allosteric activator that binds the assembly complex and stimulates the cysteine desulfurase and iron-sulfur cluster assembly activities. FXN depletion causes loss of activity of iron-sulfur-dependent enzymes and the development of the neurodegenerative disease Friedreich's ataxia. Recently, a mutation that suppressed the loss of the FXN homolog in Saccharomyces cerevisiae was identified that encodes an amino acid substitution equivalent to the human variant ISCU2 M140I. Here, we developed iron-sulfur cluster synthesis and transfer functional assays and determined that the human ISCU2 M140I variant can substitute for FXN in accelerating the rate of iron-sulfur cluster formation on the monothiol glutaredoxin (GRX5) acceptor protein. Incorporation of both FXN and the M140I substitution had an additive effect, suggesting an acceleration of distinct steps in iron-sulfur cluster biogenesis. In contrast to the canonical role of FXN in stimulating the formation of [2Fe-2S]-ISCU2 intermediates, we found here that the M140I substitution in ISCU2 promotes the transfer of iron-sulfur clusters to GRX5. Together, these results reveal an unexpected mechanism that replaces FXN-based stimulation of the iron-sulfur cluster biosynthetic pathway and suggest new strategies to overcome the loss of cellular FXN that may be relevant to the development of therapeutics for Friedreich's ataxia.
Assuntos
Ataxia de Friedreich/patologia , Proteínas de Ligação ao Ferro/metabolismo , Regulação Alostérica , Liases de Carbono-Enxofre/metabolismo , Ataxia de Friedreich/metabolismo , Glutarredoxinas/metabolismo , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Ligação Proteica , FrataxinaRESUMO
Microorganisms have lifestyles and metabolism adapted to environmental niches, which can be very broad or highly restricted. Molecular oxygen (O2) is currently variably present in microenvironments and has driven adaptation and microbial differentiation over the course of evolution on Earth. Obligate anaerobes use enzymes and cofactors susceptible to low levels of O2 and are restricted to O2-free environments, whereas aerobes typically take advantage of O2 as a reactant in many biochemical pathways and may require O2 for essential biochemical reactions. In this Perspective, we focus on analogous enzymes found in tetrapyrrole biosynthesis, modification, and degradation that are catalyzed by O2-sensitive radical S-adenosylmethionine (SAM) enzymes and by O2-dependent metalloenzymes. We showcase four transformations for which aerobic organisms use O2 as a cosubstrate but anaerobic organisms do not. These reactions include oxidative decarboxylation, methyl and methylene oxidation, ring formation, and ring cleavage. Furthermore, we highlight biochemically uncharacterized enzymes implicated in reactions that resemble those catalyzed by the parallel aerobic and anaerobic enzymes. Intriguingly, several of these reactions require insertion of an oxygen atom into the substrate, which in aerobic enzymes is facilitated by activation of O2 but in anaerobic organisms requires an alternative mechanism.
Assuntos
Enzimas/química , Enzimas/metabolismo , S-Adenosilmetionina/metabolismo , Tetrapirróis/metabolismo , Aerobiose , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catálise , Clorofila/biossíntese , Coproporfirinogênio Oxidase/química , Coproporfirinogênio Oxidase/metabolismo , Descarboxilação , Heme/metabolismo , Oxirredução , Oxigênio/metabolismo , Porfirinas/biossíntese , Porfirinas/química , Tetrapirróis/biossíntese , Tetrapirróis/químicaRESUMO
HD domain phosphohydrolase enzymes are characterized by a conserved set of histidine and aspartate residues that coordinate an active site metallocenter. Despite the important roles these enzymes play in nucleotide metabolism and signal transduction, few have been both biochemically and structurally characterized. Here, we present X-ray crystal structures and biochemical characterization of the Bacillus megaterium HD domain phosphohydrolase OxsA, involved in the biosynthesis of the antitumor, antiviral, and antibacterial compound oxetanocin-A. These studies reveal a previously uncharacterized reaction for this family; OxsA catalyzes the conversion of a triphosphorylated compound into a nucleoside, releasing one molecule of inorganic phosphate at a time. Remarkably, this functionality is a result of the OxsA active site, which based on structural and kinetic analyses has been tailored to bind the small, four-membered ring of oxetanocin-A over larger substrates. Furthermore, our OxsA structures show an active site that switches from a dinuclear to a mononuclear metal center as phosphates are eliminated from substrate.
Assuntos
Adenina/análogos & derivados , Bacillus megaterium/enzimologia , Monoéster Fosfórico Hidrolases/química , Conformação Proteica , Adenina/biossíntese , Adenina/química , Ácido Aspártico/química , Ácido Aspártico/genética , Bacillus megaterium/química , Sítios de Ligação , Catálise , Domínio Catalítico/genética , Cristalografia por Raios X , Histidina/química , Histidina/genética , Cinética , Monoéster Fosfórico Hidrolases/genética , Transdução de Sinais , Especificidade por SubstratoRESUMO
Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25-50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the availability of advanced detectors, and the incorporation of spectroscopic equipment at a number of synchrotron beamlines, have yielded exciting developments in metalloprotein structure determination. Here we will present results on the advantageous uses of metals in metalloprotein crystallography, including using metallocofactors to obtain phasing information, using K-edge X-ray absorption spectroscopy to identify metals coordinated in metalloprotein crystals, and using UV-vis spectroscopy on crystals to probe the enzymatic activity of the crystallized protein.
Assuntos
Metaloproteínas/química , Cristalografia por Raios X , Conformação Proteica , Espectroscopia por Absorção de Raios XRESUMO
Iron-sulfur clusters are ubiquitous protein cofactors with critical cellular functions. The mitochondrial Fe-S assembly complex, which consists of the cysteine desulfurase NFS1 and its accessory protein (ISD11), the Fe-S assembly protein (ISCU2), and frataxin (FXN), converts substrates l-cysteine, ferrous iron, and electrons into Fe-S clusters. The physiological function of FXN has received a tremendous amount of attention since the discovery that its loss is directly linked to the neurodegenerative disease Friedreich's ataxia. Previous in vitro results revealed a role for human FXN in activating the cysteine desulfurase and Fe-S cluster biosynthesis activities of the Fe-S assembly complex. Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe-S clusters. Additional mutagenesis, enzyme kinetic, UV-visible, and circular dichroism spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN activation, whereas C35, C61, and C104 are all essential for Fe-S cluster formation on the assembly complex. These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe-S cluster biosynthesis, and further support an allosteric regulator role for FXN. Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe-S cluster biosynthesis.
Assuntos
Proteínas de Ligação ao Ferro/química , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Enxofre/química , Cisteína/química , Humanos , Proteínas de Ligação ao Ferro/fisiologia , Proteínas Ferro-Enxofre/biossíntese , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/fisiologia , Ligação Proteica/fisiologia , Especificidade por Substrato/fisiologia , Enxofre/metabolismo , FrataxinaRESUMO
The circadian rhythms exhibited in the cyanobacterium Synechococcus elongatus are generated by an oscillator comprised of the proteins KaiA, KaiB, and KaiC. An external signal that commonly affects the circadian clock is light. Previously, we reported that the bacteriophytochrome-like protein CikA passes environmental signals to the oscillator by directly binding a quinone and using cellular redox state as a measure of light in this photosynthetic organism. Here, we report that KaiA also binds the quinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), and the oxidized form of DBMIB, but not its reduced form, decreases the stability of KaiA in vivo, causes multimerization in vitro, and blocks KaiA stimulation of KaiC phosphorylation, which is central to circadian oscillation. Our data suggest that KaiA directly senses environmental signals as changes in redox state and modulates the circadian clock.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/química , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Synechococcus/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Ritmo Circadiano/fisiologia , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Dibromotimoquinona/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Fosforilação , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Multimerização Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Synechococcus/genéticaRESUMO
Enzyme engineering plays a central role in the development of biocatalysts for biotechnology, chemical and pharmaceutical manufacturing, and environmental remediation. Rational design of proteins has historically relied on targeting active site residues to confer a protein with desirable catalytic properties. However, additional "hotspots" are also known to exist beyond the active site. Structural elements such as subunit-subunit interactions, entrance tunnels, and flexible loops influence enzyme catalysis and serve as potential "hotspots" for engineering. For the Rieske oxygenases, which use a Rieske cluster and mononuclear iron center to catalyze a challenging set of reactions, these outside of the active site regions are increasingly being shown to drive catalytic outcomes. Therefore, here, we highlight recent work on structurally characterized Rieske oxygenases that implicates architectural pieces inside and outside of the active site as key dictators of catalysis, and we suggest that these features may warrant attention in efforts aimed at Rieske oxygenase engineering.
Assuntos
Oxigenases , Oxigenases/química , Domínio Catalítico , CatáliseRESUMO
Rieske oxygenases use a Rieske-type [2Fe-2S] cluster and a mononuclear iron center to initiate a range of chemical transformations. However, few details exist regarding how this catalytic scaffold can be predictively tuned to catalyze divergent reactions. Therefore, in this work, using a combination of structural analyses, as well as substrate and rational protein-based engineering campaigns, we elucidate the architectural trends that govern catalytic outcome in the Rieske monooxygenase TsaM. We identify structural features that permit a substrate to be functionalized by TsaM and pinpoint active-site residues that can be targeted to manipulate reactivity. Exploiting these findings allowed for custom tuning of TsaM reactivity: substrates are identified that support divergent TsaM-catalyzed reactions and variants are created that exclusively catalyze dioxygenation or sequential monooxygenation chemistry. Importantly, we further leverage these trends to tune the reactivity of additional monooxygenase and dioxygenase enzymes, and thereby provide strategies to custom tune Rieske oxygenase reaction outcomes.
Assuntos
Dioxigenases , Oxigenases , Oxigenases de Função Mista , Catálise , CulturaRESUMO
Human frataxin (FXN) has been intensively studied since the discovery that the FXN gene is associated with the neurodegenerative disease Friedreich's ataxia. Human FXN is a component of the NFS1-ISD11-ISCU2-FXN (SDUF) core Fe-S assembly complex and activates the cysteine desulfurase and Fe-S cluster biosynthesis reactions. In contrast, the Escherichia coli FXN homologue CyaY inhibits Fe-S cluster biosynthesis. To resolve this discrepancy, enzyme kinetic experiments were performed for the human and E. coli systems in which analogous cysteine desulfurase, Fe-S assembly scaffold, and frataxin components were interchanged. Surprisingly, our results reveal that activation or inhibition by the frataxin homologue is determined by which cysteine desulfurase is present and not by the identity of the frataxin homologue. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologues. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule.
Assuntos
Proteínas de Ligação ao Ferro/metabolismo , Ferro/metabolismo , Enxofre/metabolismo , Liases de Carbono-Enxofre/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas de Ligação ao Ferro/química , Proteínas Reguladoras de Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Homologia de Sequência de Aminoácidos , FrataxinaRESUMO
The cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster, SAM, and Cbl to carry out remarkable catalytic feats in a large number of biosynthetic pathways. However, despite the abundance of annotated Cbl-dependent radical SAM enzymes, relatively few molecular details exist regarding how these enzymes function. Traditionally, challenges associated with purifying and reconstituting Cbl-dependent radical SAM enzymes have hindered biochemical studies aimed at elucidating the structures and mechanisms of these enzymes. Herein, we describe a bottom-up approach that was used to crystallize OxsB, learn about the overall architecture of a Cbl-dependent radical SAM enzyme, and facilitate mechanistic studies. We report lessons learned from the crystallization of different states of OxsB, including the apo-, selenomethionine (SeMet)-labeled, and fully reconstituted form of OxsB that has a [4Fe-4S] cluster, SAM, and Cbl bound. Further, we suggest that, when appropriate, this bottom-up method can be used to facilitate studies on enzymes in this class for which there are challenges associated with purifying and reconstituting the active enzyme.
Assuntos
Proteínas Ferro-Enxofre , S-Adenosilmetionina , Vias Biossintéticas , Proteínas Ferro-Enxofre/metabolismo , S-Adenosilmetionina/metabolismo , Vitamina B 12/metabolismoRESUMO
The members of the radical S-adenosylmethionine (SAM) enzyme superfamily are responsible for catalyzing a diverse set of reactions in a multitude of biosynthetic pathways. Many members of this superfamily accomplish their transformations using the catalytic power of a 5'-deoxyadenosyl radical (5'-dAdoâ¢), but there are also enzymes within this superfamily that bind auxiliary cofactors and extend the catalytic repertoire of SAM. In particular, the cobalamin (Cbl)-dependent class synergistically uses Cbl to facilitate challenging methylation and radical rearrangement reactions. Despite identification of this class by Sofia et al. 20 years ago, the low sequence identity between members has led to difficulty in predicting function of uncharacterized members, pinpointing catalytic residues, and elucidating reaction mechanisms. Here, we capitalize on the three recent structures of Cbl-dependent radical SAM enzymes that use common cofactors to facilitate ring contraction as well as radical-based and non-radical-based methylation reactions. With these three structures as a framework, we describe how the Cbl-dependent radical SAM enzymes repurpose the traditional SAM- and Cbl-binding motifs to form an active site where both Cbl and SAM can participate in catalysis. In addition, we describe how, in some cases, the classic SAM- and Cbl-binding motifs support the diverse functionality of this enzyme class, and finally, we define new motifs that are characteristic of Cbl-dependent radical SAM enzymes.