RESUMO
Technologically critical rare-earth elements are notoriously difficult to separate, owing to their subtle differences in ionic radius and coordination number1-3. The natural lanthanide-binding protein lanmodulin (LanM)4,5 is a sustainable alternative to conventional solvent-extraction-based separation6. Here we characterize a new LanM, from Hansschlegelia quercus (Hans-LanM), with an oligomeric state sensitive to rare-earth ionic radius, the lanthanum(III)-induced dimer being >100-fold tighter than the dysprosium(III)-induced dimer. X-ray crystal structures illustrate how picometre-scale differences in radius between lanthanum(III) and dysprosium(III) are propagated to Hans-LanM's quaternary structure through a carboxylate shift that rearranges a second-sphere hydrogen-bonding network. Comparison to the prototypal LanM from Methylorubrum extorquens reveals distinct metal coordination strategies, rationalizing Hans-LanM's greater selectivity within the rare-earth elements. Finally, structure-guided mutagenesis of a key residue at the Hans-LanM dimer interface modulates dimerization in solution and enables single-stage, column-based separation of a neodymium(III)/dysprosium(III) mixture to >98% individual element purities. This work showcases the natural diversity of selective lanthanide recognition motifs, and it reveals rare-earth-sensitive dimerization as a biological principle by which to tune the performance of biomolecule-based separation processes.
Assuntos
Proteínas de Bactérias , Elementos da Série dos Lantanídeos , Lantânio , Multimerização Proteica , Disprósio/química , Disprósio/isolamento & purificação , Íons/química , Elementos da Série dos Lantanídeos/química , Elementos da Série dos Lantanídeos/isolamento & purificação , Lantânio/química , Neodímio/química , Neodímio/isolamento & purificação , Methylocystaceae , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Estrutura Quaternária de ProteínaRESUMO
Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.
Assuntos
Carbapenêmicos/biossíntese , Metiltransferases/química , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Streptomyces/enzimologia , Tienamicinas/biossíntese , Vitamina B 12/metabolismo , Sítios de Ligação , Biocatálise , Coenzimas/metabolismo , Cristalografia por Raios X , Cinética , Metilação , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Streptomyces/metabolismo , Inibidores de beta-Lactamases/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismoRESUMO
Archaea synthesize isoprenoid-based ether-linked membrane lipids, which enable them to withstand extreme environmental conditions, such as high temperatures, high salinity, and low or high pH values1-5. In some archaea, such as Methanocaldococcus jannaschii, these lipids are further modified by forming carbon-carbon bonds between the termini of two lipid tails within one glycerophospholipid to generate the macrocyclic archaeol or forming two carbon-carbon bonds between the termini of two lipid tails from two glycerophospholipids to generate the macrocycle glycerol dibiphytanyl glycerol tetraether (GDGT)1,2. GDGT contains two 40-carbon lipid chains (biphytanyl chains) that span both leaflets of the membrane, providing enhanced stability to extreme conditions. How these specialized lipids are formed has puzzled scientists for decades. The reaction necessitates the coupling of two completely inert sp3-hybridized carbon centres, which, to our knowledge, has not been observed in nature. Here we show that the gene product of mj0619 from M. jannaschii, which encodes a radical S-adenosylmethionine enzyme, is responsible for biphytanyl chain formation during synthesis of both the macrocyclic archaeol and GDGT membrane lipids6. Structures of the enzyme show the presence of four metallocofactors: three [Fe4S4] clusters and one mononuclear rubredoxin-like iron ion. In vitro mechanistic studies show that Csp3-Csp3 bond formation takes place on fully saturated archaeal lipid substrates and involves an intermediate bond between the substrate carbon and a sulfur of one of the [Fe4S4] clusters. Our results not only establish the biosynthetic route for tetraether formation but also improve the use of GDGT in GDGT-based paleoclimatology indices7-10.
Assuntos
Proteínas Arqueais , Éteres de Glicerila , Lipídeos de Membrana , Methanocaldococcus , Proteínas Arqueais/química , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Carbono/química , Carbono/metabolismo , Glicerol/química , Glicerol/metabolismo , Éteres de Glicerila/química , Éteres de Glicerila/metabolismo , Lipídeos de Membrana/biossíntese , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Methanocaldococcus/química , Methanocaldococcus/enzimologia , Methanocaldococcus/metabolismo , S-Adenosilmetionina/metabolismo , Terpenos/química , Terpenos/metabolismoRESUMO
Small molecules containing the N-nitroso group, such as the bacterial natural product streptozotocin, are prominent carcinogens1,2 and important cancer chemotherapeutics3,4. Despite the considerable importance of this functional group to human health, enzymes dedicated to the assembly of the N-nitroso unit have not been identified. Here we show that SznF, a metalloenzyme from the biosynthesis of streptozotocin, catalyses an oxidative rearrangement of the guanidine group of Nω-methyl-L-arginine to generate an N-nitrosourea product. Structural characterization and mutagenesis of SznF reveal two separate active sites that promote distinct steps in this transformation using different iron-containing metallocofactors. This biosynthetic reaction, which has little precedent in enzymology or organic synthesis, expands the catalytic capabilities of non-haem-iron-dependent enzymes to include N-N bond formation. We find that biosynthetic gene clusters that encode SznF homologues are widely distributed among bacteria-including environmental organisms, plant symbionts and human pathogens-which suggests an unexpectedly diverse and uncharacterized microbial reservoir of bioactive N-nitroso metabolites.
Assuntos
Metaloproteínas/metabolismo , Estreptozocina/biossíntese , Estreptozocina/química , Arginina/análogos & derivados , Domínio Catalítico/genética , Coenzimas/metabolismo , Cristalografia por Raios X , Guanidina/metabolismo , Ferro/metabolismo , Metaloproteínas/química , Metaloproteínas/genética , Modelos Moleculares , Família Multigênica , Compostos de Nitrosoureia/metabolismo , Streptomyces/enzimologia , Streptomyces/genéticaRESUMO
Hyoscyamine 6ß-hydroxylase (H6H) is an iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase that produces the prolifically administered antinausea drug, scopolamine. After its namesake hydroxylation reaction, H6H then couples the newly installed C6 oxygen to C7 to produce the drug's epoxide functionality. Oxoiron(IV) (ferryl) intermediates initiate both reactions by cleaving C-H bonds, but it remains unclear how the enzyme switches the target site and promotes (C6)O-C7 coupling in preference to C7 hydroxylation in the second step. In one possible epoxidation mechanism, the C6 oxygen wouldâanalogously to mechanisms proposed for the Fe/2OG halogenases and, in our more recent study, N-acetylnorloline synthase (LolO)âcoordinate as alkoxide to the C7-H-cleaving ferryl intermediate to enable alkoxyl coupling to the ensuing C7 radical. Here, we provide structural and kinetic evidence that H6H does not employ substrate coordination or repositioning for the epoxidation step but instead exploits the distinct spatial dependencies of competitive C-H cleavage (C6 vs C7) and C-O-coupling (oxygen rebound vs cyclization) steps to promote the two-step sequence. Structural comparisons of ferryl-mimicking vanadyl complexes of wild-type H6H and a variant that preferentially 7-hydroxylates instead of epoxidizing 6ß-hydroxyhyoscyamine suggest that a modest (â¼10°) shift in the Fe-O-H(C7) approach angle is sufficient to change the outcome. The 7-hydroxylation:epoxidation partition ratios of both proteins increase more than 5-fold in 2H2O, reflecting an epoxidation-specific requirement for cleavage of the alcohol O-H bond, which, unlike in the LolO oxacyclization, is not accomplished by iron coordination in advance of C-H cleavage.
RESUMO
In biosynthesis of the pancreatic cancer drug streptozotocin, the tridomain nonheme-iron oxygenase SznF hydroxylates Nδ and Nω' of Nω-methyl-l-arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the monoiron cofactor in the enzyme's C-terminal cupin domain, which promotes the final rearrangement, but exhibited disorder and minimal metal occupancy in the site of the proposed diiron cofactor in the N-hydroxylating heme-oxygenase-like (HO-like) central domain. We leveraged our recent observation that the N-oxygenating µ-peroxodiiron(III/III) intermediate can form in the HO-like domain after the apo protein self-assembles its diiron(II/II) cofactor to solve structures of SznF with both of its iron cofactors bound. These structures of a biochemically validated member of the emerging heme-oxygenase-like diiron oxidase and oxygenase (HDO) superfamily with intact diiron cofactor reveal both the large-scale conformational change required to assemble the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability-a trait shared by the other validated HDOs. During cofactor (dis)assembly, a ligand-harboring core helix dynamically (un)folds. The diiron cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulations. The additional carboxylate ligand is conserved in another N-oxygenating HDO but not in two HDOs that cleave carbon-hydrogen and carbon-carbon bonds to install olefins. Among â¼9,600 sequences identified bioinformatically as members of the emerging HDO superfamily, â¼25% conserve this additional carboxylate residue and are thus tentatively assigned as N-oxygenases.
Assuntos
Heme Oxigenase (Desciclizante)/ultraestrutura , Ferroproteínas não Heme/ultraestrutura , Oxigenases/ultraestrutura , Estreptozocina/química , Catálise/efeitos dos fármacos , Cristalografia por Raios X , Heme Oxigenase (Desciclizante)/química , Humanos , Ligantes , Compostos de Nitrosoureia/toxicidade , Ferroproteínas não Heme/química , Oxirredução , Oxigênio/química , Oxigenases/química , Neoplasias Pancreáticas/induzido quimicamente , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Conformação Proteica/efeitos dos fármacos , Domínios Proteicos/genética , Estreptozocina/toxicidadeRESUMO
An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl- or Br-), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three nongaseous substrates must bind to activate the enzyme's Fe(II) cofactor for efficient capture of O2. Halide, 2OG, and (lastly) O2 all coordinate directly to the cofactor to initiate its conversion to a cis-halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (Hâ¢) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of the l-lysine 4-chlorinase, BesD. After addition of 2OG, subsequent coordination of the halide to the cofactor and binding of cationic l-Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon the addition of O2 does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide and l-Lys. The surprising lability of the BesDâ¢[Fe(IV)=O]â¢Clâ¢succinateâ¢l-Lys complex engenders pathways for decay of the haloferryl intermediate that do not result in l-Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply (i) that BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage between l-Lys binding and chloride coordination following the loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.
Assuntos
Cloretos , Lisina , Oxigenases de Função Mista/química , Ferro/química , Oxirredução , Oxigênio/químicaRESUMO
The enzyme BesC from the ß-ethynyl-l-serine biosynthetic pathway in Streptomyces cattleya fragments 4-chloro-l-lysine (produced from l-Lysine by BesD) to ammonia, formaldehyde, and 4-chloro-l-allylglycine and can analogously fragment l-Lys itself. BesC belongs to the emerging family of O2-activating non-heme-diiron enzymes with the "heme-oxygenase-like" protein fold (HDOs). Here, we show that the binding of l-Lys or an analogue triggers capture of O2 by the protein's diiron(II) cofactor to form a blue µ-peroxodiiron(III) intermediate analogous to those previously characterized in two other HDOs, the olefin-installing fatty acid decarboxylase, UndA, and the guanidino-N-oxygenase domain of SznF. The â¼5- and â¼30-fold faster decay of the intermediate in reactions with 4-thia-l-Lys and (4RS)-chloro-dl-lysine than in the reaction with l-Lys itself and the primary deuterium kinetic isotope effects (D-KIEs) on decay of the intermediate and production of l-allylglycine in the reaction with 4,4,5,5-[2H4]-l-Lys suggest that the peroxide intermediate or a reversibly connected successor complex abstracts a hydrogen atom from C4 to enable olefin formation. Surprisingly, the sluggish substrate l-Lys can dissociate after triggering intermediate formation, thereby allowing one of the better substrates to bind and react. The structure of apo BesC and the demonstrated linkage between Fe(II) and substrate binding suggest that the triggering event involves an induced ordering of ligand-providing helix 3 (α3) of the conditionally stable HDO core. As previously suggested for SznF, the dynamic α3 also likely initiates the spontaneous degradation of the diiron(III) product cluster after decay of the peroxide intermediate, a trait emerging as characteristic of the nascent HDO family.
Assuntos
Heme Oxigenase (Desciclizante) , Oxirredutases , Alilglicina , Heme , Lisina , Oxirredutases/metabolismo , Oxigênio/metabolismo , Oxigenases/química , PeróxidosRESUMO
In most organisms, transition metal ions are necessary cofactors of ribonucleotide reductase (RNR), the enzyme responsible for biosynthesis of the 2'-deoxynucleotide building blocks of DNA. The metal ion generates an oxidant for an active site cysteine (Cys), yielding a thiyl radical that is necessary for initiation of catalysis in all RNRs. Class I enzymes, widespread in eukaryotes and aerobic microbes, share a common requirement for dioxygen in assembly of the active Cys oxidant and a unique quaternary structure, in which the metallo- or radical-cofactor is found in a separate subunit, ß, from the catalytic α subunit. The first class I RNRs, the class Ia enzymes, discovered and characterized more than 30 years ago, were found to use a diiron(III)-tyrosyl-radical Cys oxidant. Although class Ia RNRs have historically served as the model for understanding enzyme mechanism and function, more recently, remarkably diverse bioinorganic and radical cofactors have been discovered in class I RNRs from pathogenic microbes. These enzymes use alternative transition metal ions, such as manganese, or posttranslationally installed tyrosyl radicals for initiation of ribonucleotide reduction. Here we summarize the recent progress in discovery and characterization of novel class I RNR radical-initiating cofactors, their mechanisms of assembly, and how they might function in the context of the active class I holoenzyme complex.
Assuntos
Coenzimas , Metais , Ribonucleotídeo Redutases , Animais , Catálise , Domínio Catalítico , Coenzimas/química , Coenzimas/classificação , Coenzimas/metabolismo , Humanos , Metais/química , Metais/metabolismo , Oxirredução , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/metabolismoRESUMO
Speckle-type POZ protein (SPOP) is a ubiquitin ligase adaptor that binds substrate proteins and facilitates their proteasomal degradation. Most SPOP substrates present multiple SPOP-binding (SB) motifs and undergo liquid-liquid phase separation with SPOP. Pancreatic and duodenal homeobox 1 (Pdx1), an insulin transcription factor, is downregulated by interaction with SPOP. Unlike other substrates, only one SB motif has previously been reported within the Pdx1 C-terminal intrinsically disordered region (Pdx1-C). Given this difference, we aimed to determine the specific mode of interaction of Pdx1 with SPOP and how it is similar or different to that of other SPOP substrates. Here, we identify a second SB motif in Pdx1-C, but still find that the resulting moderate valency is insufficient to support phase separation with SPOP in cells. Although Pdx1 does not phase separate with SPOP, Pdx1 and SPOP interaction prompts SPOP relocalization from nuclear speckles to the diffuse nucleoplasm. Accordingly, we find that SPOP-mediated ubiquitination activity of Pdx1 occurs in the nucleoplasm and that highly efficient Pdx1 turnover requires both SB motifs. Our results suggest that the subnuclear localization of SPOP-substrate interactions and substrate ubiquitination may be directed by the properties of the substrate itself.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitinação , Motivos de Aminoácidos , Proteínas de Homeodomínio/metabolismo , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Ligação Proteica , Transporte Proteico , Proteínas Repressoras/química , Transativadores/metabolismoRESUMO
Ethylene-forming enzyme (EFE) is an ambifunctional iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase. In its major (EF) reaction, it converts carbons 1, 2, and 5 of 2OG to CO2 and carbons 3 and 4 to ethylene, a four-electron oxidation drastically different from the simpler decarboxylation of 2OG to succinate mediated by all other Fe/2OG enzymes. EFE also catalyzes a minor reaction, in which the normal decarboxylation is coupled to oxidation of l-arginine (a required activator for the EF pathway), resulting in its conversion to l-glutamate semialdehyde and guanidine. Here we show that, consistent with precedent, the l-Arg-oxidation (RO) pathway proceeds via an iron(IV)-oxo (ferryl) intermediate. Use of 5,5-[2H2]-l-Arg slows decay of the ferryl complex by >16-fold, implying that RO is initiated by hydrogen-atom transfer (HAT) from C5. That this large substrate deuterium kinetic isotope effect has no impact on the EF:RO partition ratio implies that the same ferryl intermediate cannot be on the EF pathway; the pathways must diverge earlier. Consistent with this conclusion, the variant enzyme bearing the Asp191Glu ligand substitution accumulates â¼4 times as much of the ferryl complex as the wild-type enzyme and exhibits a â¼40-fold diminished EF:RO partition ratio. The selective detriment of this nearly conservative substitution to the EF pathway implies that it has unusually stringent stereoelectronic requirements. An active-site, like-charge guanidinium pair, which involves the l-Arg substrate/activator and is unique to EFE among four crystallographically characterized l-Arg-modifying Fe/2OG oxygenases, may serve to selectively stabilize the transition state leading to the unique EF branch.
Assuntos
Arginina/química , Ferro/química , Ácidos Cetoglutáricos/metabolismo , Oxigenases/metabolismo , Modelos Moleculares , Oxirredução , Oxigenases/química , Conformação ProteicaRESUMO
The high fidelity of DNA replication and repair is attributable, in part, to the allosteric regulation of ribonucleotide reductases (RNRs) that maintains proper deoxynucleotide pool sizes and ratios in vivo. In class Ia RNRs, ATP (stimulatory) and dATP (inhibitory) regulate activity by binding to the ATP-cone domain at the N terminus of the large α subunit and altering the enzyme's quaternary structure. Class Ib RNRs, in contrast, have a partial cone domain and have generally been found to be insensitive to dATP inhibition. An exception is the Bacillus subtilis Ib RNR, which we recently reported to be inhibited by physiological concentrations of dATP. Here, we demonstrate that the α subunit of this RNR contains tightly bound deoxyadenosine 5'-monophosphate (dAMP) in its N-terminal domain and that dATP inhibition of CDP reduction is enhanced by its presence. X-ray crystallography reveals a previously unobserved (noncanonical) α2 dimer with its entire interface composed of the partial N-terminal cone domains, each binding a dAMP molecule. Using small-angle X-ray scattering (SAXS), we show that this noncanonical α2 dimer is the predominant form of the dAMP-bound α in solution and further show that addition of dATP leads to the formation of larger oligomers. Based on this information, we propose a model to describe the mechanism by which the noncanonical α2 inhibits the activity of the B. subtilis Ib RNR in a dATP- and dAMP-dependent manner.
Assuntos
Bacillus subtilis/enzimologia , Nucleotídeos de Desoxiadenina/metabolismo , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Regulação Alostérica , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Nucleotídeos de Desoxiadenina/química , Ligantes , Ligação Proteica , Conformação Proteica , Ribonucleotídeo Redutases/genética , Espalhamento a Baixo Ângulo , Especificidade por SubstratoRESUMO
All cells obtain 2'-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, ß; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cysâ¢). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys⢠in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPAâ¢) in ß. The three-electron oxidation producing DOPA⢠occurs in Escherichia coli only if the ß is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.
Assuntos
Di-Hidroxifenilalanina/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Radicais Livres/química , Ribonucleotídeo Redutases/química , Tirosina/química , Di-Hidroxifenilalanina/metabolismo , Proteínas de Escherichia coli/metabolismo , Radicais Livres/metabolismo , Ribonucleotídeo Redutases/metabolismo , Tirosina/metabolismoRESUMO
The alkylating warhead of the pancreatic cancer drug streptozotocin (SZN) contains an N-nitrosourea moiety constructed from Nω-methyl-l-arginine (l-NMA) by the multi-domain metalloenzyme SznF. The enzyme's central heme-oxygenase-like (HO-like) domain sequentially hydroxylates Nδ and Nω' of l-NMA. Its C-terminal cupin domain then rearranges the triply modified arginine to Nδ-hydroxy-Nω-methyl-Nω-nitroso-l-citrulline, the proposed donor of the functional pharmacophore. Here we show that the HO-like domain of SznF can bind Fe(II) and use it to capture O2, forming a peroxo-Fe2(III/III) intermediate. This intermediate has absorption- and Mössbauer-spectroscopic features similar to those of complexes previously trapped in ferritin-like diiron oxidases and oxygenases (FDOs) and, more recently, the HO-like fatty acid oxidase UndA. The SznF peroxo-Fe2(III/III) complex is an intermediate in both hydroxylation steps, as shown by the concentration-dependent acceleration of its decay upon exposure to either l-NMA or Nδ-hydroxy-Nω-methyl-l-Arg (l-HMA). The Fe2(III/III) cluster produced upon decay of the intermediate has a small Mössbauer quadrupole splitting parameter, implying that, unlike the corresponding product states of many FDOs, it lacks an oxo-bridge. The subsequent decomposition of the product cluster to one or more paramagnetic Fe(III) species over several hours explains why SznF was previously purified and crystallographically characterized without its cofactor. Programmed instability of the oxidized form of the cofactor appears to be a unifying characteristic of the emerging superfamily of HO-like diiron oxidases and oxygenases (HDOs).
Assuntos
Proteínas de Bactérias/metabolismo , Compostos Férricos/metabolismo , Metaloproteínas/metabolismo , Compostos de Nitrosoureia/metabolismo , Estreptozocina/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Compostos Férricos/química , Hidroxilação , Metaloproteínas/química , Metaloproteínas/isolamento & purificação , Modelos Moleculares , Estrutura Molecular , Compostos de Nitrosoureia/química , Streptomyces/enzimologia , Estreptozocina/químicaRESUMO
X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.
Assuntos
Cristalografia por Raios X/métodos , Lasers , Acústica , Complexo de Proteína do Fotossistema II/química , Fitocromo/química , Ribonucleotídeo Redutases/química , Espectrometria por Raios X/métodosRESUMO
Iron(II)- and 2-(oxo)-glutarate-dependent (Fe/2OG) oxygenases catalyze a diverse array of oxidation reactions via a common iron(IV)-oxo (ferryl) intermediate. Although the intermediate has been characterized spectroscopically, its short lifetime has precluded crystallograhic characterization. In solution, the ferryl was first observed directly in the archetypal Fe/2OG hydroxylase, taurine:2OG dioxygenase (TauD). Here, we substitute the iron cofactor of TauD with the stable vanadium(IV)-oxo (vanadyl) ion to obtain crystal structures mimicking the key ferryl complex. Intriguingly, whereas the structure of the TauD·(VIV-oxo)·succinate·taurine complex exhibits the expected orientation of the V≡O bond-trans to the His255 ligand and toward the C-H bond to be cleaved, in what has been termed the in-line configuration-the TauD·(VIV-oxo) binary complex is best modeled with its oxo ligand trans to Asp101. This off-line-like configuration is similar to one recently posited as a means to avoid hydroxylation in Fe/2OG enzymes that direct other outcomes, though neither has been visualized in an Fe/2OG structure to date. Whereas an off-line (trans to the proximal His) or off-line-like (trans to the carboxylate ligand) ferryl is unlikely to be important in the hydroxylation reaction of TauD, the observation that the ferryl may deviate from an in-line orientation in the absence of the primary substrate may explain the enzyme's mysterious self-hydroxylation behavior, should the oxo ligand lie trans to His99. This finding reinforces the potential for analogous functional off-line oxo configurations in halogenases, desaturases, and/or cyclases.
Assuntos
Ferro/química , Oxigenases de Função Mista/química , Mimetismo Molecular , Vanadatos/química , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Escherichia coli/química , Ligação de Hidrogênio , Hidroxilação , Oxigenases de Função Mista/isolamento & purificação , Estrutura Secundária de Proteína , Ácido Succínico/química , Taurina/química , Espectroscopia por Absorção de Raios XRESUMO
Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate ß subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a MnII2 cluster by free superoxide to yield a metal-based MnIIIMnIV oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia-c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae ( Fj) and Actinobacillus ureae ( Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP-cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive ß subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher-order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.
Assuntos
Domínio Catalítico , Desoxirribonucleotídeos/química , Conformação Proteica , Ribonucleotídeo Redutases/química , Actinobacillus/enzimologia , Actinobacillus/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Biocatálise , Cristalografia por Raios X , Desoxirribonucleotídeos/biossíntese , Desoxirribonucleotídeos/genética , Flavobacterium/enzimologia , Flavobacterium/genética , Modelos Moleculares , Filogenia , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/genética , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Difração de Raios XRESUMO
The assignment of biochemical functions to hypothetical proteins is challenged by functional diversification within many protein structural superfamilies. This diversification, which is particularly common for metalloenzymes, renders functional annotations that are founded solely on sequence and domain similarities unreliable and often erroneous. Definitive biochemical characterization to delineate functional subgroups within these superfamilies will aid in improving bioinformatic approaches for functional annotation. We describe here the structural and functional characterization of two non-heme-iron oxygenases, TmpA and TmpB, which are encoded by a genomically clustered pair of genes found in more than 350 species of bacteria. TmpA and TmpB are functional homologues of a pair of enzymes (PhnY and PhnZ) that degrade 2-aminoethylphosphonate but instead act on its naturally occurring, quaternary ammonium analogue, 2-(trimethylammonio)ethylphosphonate (TMAEP). TmpA, an iron(II)- and 2-(oxo)glutarate-dependent oxygenase misannotated as a γ-butyrobetaine (γbb) hydroxylase, shows no activity toward γbb but efficiently hydroxylates TMAEP. The product, ( R)-1-hydroxy-2-(trimethylammonio)ethylphosphonate [( R)-OH-TMAEP], then serves as the substrate for the second enzyme, TmpB. By contrast to its purported phosphohydrolytic activity, TmpB is an HD-domain oxygenase that uses a mixed-valent diiron cofactor to enact oxidative cleavage of the C-P bond of its substrate, yielding glycine betaine and phosphate. The high specificities of TmpA and TmpB for their N-trimethylated substrates suggest that they have evolved specifically to degrade TMAEP, which was not previously known to be subject to microbial catabolism. This study thus adds to the growing list of known pathways through which microbes break down organophosphonates to harvest phosphorus, carbon, and nitrogen in nutrient-limited niches.
Assuntos
Ácido Aminoetilfosfônico/análogos & derivados , Proteínas de Bactérias/química , Oxigenases/química , Ácido Aminoetilfosfônico/química , Proteínas de Bactérias/genética , Escherichia coli/genética , Humanos , Ferro/química , Ácidos Cetoglutáricos/química , Organofosfonatos , Compostos Organofosforados/química , Oxirredução , Oxigenases/genética , Pseudomonas/enzimologia , Rhodobacteraceae/enzimologia , Especificidade por SubstratoRESUMO
Cfr is a radical S-adenosylmethionine (SAM) RNA methylase linked to multidrug antibiotic resistance in bacterial pathogens. It catalyzes a chemically challenging C-C bond-forming reaction to methylate C8 of A2503 (Escherichia coli numbering) of 23S rRNA during ribosome assembly. The cfr gene has been identified as a mobile genetic element in diverse bacteria and in the genome of select Bacillales and Clostridiales species. Despite the importance of Cfr, few representatives have been purified and characterized in vitro. Here we show that Cfr homologues from Bacillus amyloliquefaciens, Enterococcus faecalis, Paenibacillus lautus, and Clostridioides difficile act as C8 adenine RNA methylases in biochemical assays. C. difficile Cfr contains an additional Cys-rich C-terminal domain that binds a mononuclear Fe2+ ion in a rubredoxin-type Cys4 motif. The C-terminal domain can be truncated with minimal impact on C. difficile Cfr activity, but the rate of turnover is decreased upon disruption of the Fe2+-binding site by Zn2+ substitution or ligand mutation. These findings indicate an important purpose for the observed C-terminal iron in the native fusion protein. Bioinformatic analysis of the C. difficile Cfr Cys-rich domain shows that it is widespread (â¼1400 homologues) as a stand-alone gene in pathogenic or commensal Bacilli and Clostridia, with >10% encoded adjacent to a predicted radical SAM RNA methylase. Although the domain is not essential for in vitro C. difficile Cfr activity, the genomic co-occurrence and high abundance in the human microbiome suggest a possible functional role for a specialized rubredoxin in certain radical SAM RNA methylases that are relevant to human health.
Assuntos
Clostridioides difficile/metabolismo , Ferro/metabolismo , Filogenia , RNA/metabolismo , S-Adenosilmetionina/metabolismo , Sequência de Aminoácidos , Clostridioides difficile/genética , Metilação , Ligação Proteica/fisiologia , RNA/genéticaRESUMO
(S)-2-Hydroxypropylphosphonate [(S)-2-HPP, 1] epoxidase (HppE) reduces H2O2 at its nonheme-iron cofactor to install the oxirane "warhead" of the antibiotic fosfomycin. The net replacement of the C1 pro-R hydrogen of 1 by its C2 oxygen, with inversion of configuration at C1, yields the cis-epoxide of the drug [(1R,2S)-epoxypropylphosphonic acid (cis-Fos, 2)]. Here we show that HppE achieves â¼95% selectivity for C1 inversion and cis-epoxide formation via steric guidance of a radical-coupling mechanism. Published structures of the HppE·FeII·1 and HppE·ZnII·2 complexes reveal distinct pockets for C3 of the substrate and product and identify four hydrophobic residues-Leu120, Leu144, Phe182, and Leu193-close to C3 in one of the complexes. Replacement of Leu193 in the substrate C3 pocket with the bulkier Phe enhances stereoselectivity (cis:trans â¼99:1), whereas the Leu120Phe substitution in the product C3 pocket diminishes it (â¼82:18). Retention of C1 configuration and trans-epoxide formation become predominant with the bulk-reducing Phe182Ala substitution in the substrate C3 pocket (â¼13:87), trifluorination of C3 (â¼23:77), or both (â¼1:99). The effect of C3 trifluorination is counteracted by the more constrained substrate C3 pockets in the Leu193Phe (â¼56:44) and Leu144Phe/Leu193Phe (â¼90:10) variants. The ability of HppE to epoxidize substrate analogues bearing halogens at C3, C1, or both is inconsistent with a published hypothesis of polar cyclization via a C1 carbocation. Rather, specific enzyme-substrate contacts drive inversion of the C1 radical-as proposed in a recent computational study-to direct formation of the more potently antibacterial cis-epoxide by radicaloid C-O coupling.