RESUMO
PylB is a radical S-adenosyl-l-methionine (SAM) enzyme predicted to convert l-lysine into (3R)-3-methyl-d-ornithine, a precursor in the biosynthesis of the 22nd proteogenic amino acid pyrrolysine. This protein highly resembles that of the radical SAM tyrosine and tryptophan lyases, which activate their substrate by abstracting a H atom from the amino-nitrogen position. Here, combining in vitro assays, analytical methods, electron paramagnetic resonance spectroscopy, and theoretical methods, we demonstrated that instead, PylB activates its substrate by abstracting a H atom from the Cγ position of l-lysine to afford the radical-based ß-scission. Strikingly, we also showed that PylB catalyzes the reverse reaction, converting (3R)-3-methyl-d-ornithine into l-lysine and using catalytic amounts of the 5'-deoxyadenosyl radical. Finally, we identified significant in vitro production of 5'-thioadenosine, an unexpected shunt product that we propose to result from the quenching of the 5'-deoxyadenosyl radical species by the nearby [Fe4S4] cluster.
Assuntos
Metionina , Ornitina/análogos & derivados , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , Lisina , Racemetionina , Espectroscopia de Ressonância de Spin EletrônicaRESUMO
Naturally occurring protein nanocages like ferritin are self-assembled from multiple subunits. Because of their unique cage-like structure and biocompatibility, there is a growing interest in their biomedical use. A multipurpose and straightforward engineering approach does not exist for using nanocages to make drug-delivery systems by encapsulating hydrophilic or hydrophobic drugs and developing vaccines by surface functionalization with a protein like an antigen. Here, a versatile engineering approach is described by mimicking the HIV-1 Gap polyprotein precursor. Various PREcursors of nanoCages (PREC) are designed and created by linking two ferritin subunits via a flexible linker peptide containing a protease cleavage site. These precursors can have additional proteins at their N-terminus, and their protease cleavage generates ferritin-like nanocages named protease-induced nanocages (PINCs). It is demonstrated that PINC formation allows concurrent surface decoration with a protein and hydrophilic or hydrophobic drug encapsulation up to fourfold more than the amount achieved using other methods. The PINCs/Drug complex is stable and efficiently kills cancer cells. This work provides insight into the precursors' design rules and the mechanism of PINCs formation. The engineering approach and mechanistic insight described here will facilitate nanocages' applications in drug delivery or as a platform for making multifunctional therapeutics like mosaic vaccines.
Assuntos
Ferritinas , Humanos , Ferritinas/química , Propriedades de Superfície , HIV-1 , Interações Hidrofóbicas e Hidrofílicas , Sistemas de Liberação de Medicamentos/métodos , Nanoestruturas/química , Materiais Biomiméticos/química , Biomimética/métodosRESUMO
[FeFe]-hydrogenases efficiently catalyze the reversible oxidation of molecular hydrogen. Their prowess stems from the intricate H-cluster, combining a [Fe4 S4 ] center with a binuclear iron center ([2Fe]H ). In the latter, each iron atom is coordinated by a CO and CN ligand, connected by a CO and an azadithiolate ligand. The synthesis of this active site involves a unique multiprotein assembly, featuring radical SAM proteins HydG and HydE. HydG initiates the transformation of L-tyrosine into cyanide and carbon monoxide to generate complex B, which is subsequently transferred to HydE to continue the biosynthesis of the [2Fe]H -subcluster. Due to its instability, complex B isolation for structural or spectroscopic characterization has been elusive thus far. Nevertheless, the use of a biomimetic analogue of complex B allowed circumvention of the need for the HydG protein during in vitro functional investigations, implying a similar structure for complex B. Herein, we used the HydE protein as a nanocage to encapsulate and stabilize the complex B product generated by HydG. Using X-ray crystallography, we successfully determined its structure at 1.3â Å resolution. Furthermore, we demonstrated that complex B is directly transferred from HydG to HydE, thus not being released into the solution post-synthesis, highlighting a transient interaction between the two proteins.
Assuntos
Hidrogenase , Proteínas Ferro-Enxofre , Hidrogenase/metabolismo , Ligantes , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas/metabolismo , Ferro/química , Compostos Ferrosos/metabolismo , Proteínas Ferro-Enxofre/químicaRESUMO
Darobactin A is a ribosomally synthesized, post-translationally modified peptide (RiPP) with potent and broad-spectrum anti-Gram-negative antibiotic activity. The structure of darobactin A is characterized by an ether and C-C crosslinking. However, the specific mechanism of the crosslink formation, especially the ether crosslink, remains elusive. Here, using in vitro enzyme assays, we demonstrate that both crosslinks are formed by the DarE radical S-adenosylmethionine (SAM) enzyme in an O2-dependent manner. The relevance of the observed activity to darobactin A biosynthesis was demonstrated by proteolytic transformation of the DarE product into darobactin A. Furthermore, DarE assays in the presence of 18O2 or [18O]water demonstrated that the oxygen of the ether crosslink originates from O2 and not from water. These results demonstrate that DarE is a radical SAM enzyme that uses oxygen as a co-substrate in its physiologically relevant function. Since radical SAM enzymes are generally considered to function under anaerobic environments, the discovery of a radical SAM oxygenase represents a significant change in the paradigm and suggests that these radical SAM enzymes function in aerobic cells. Also, the study revealed that DarE catalyzes the formation of three distinct modifications on DarA; ether and C-C crosslinks and α,ß-desaturation. Based on these observations, possible mechanisms of the DarE-catalyzed reactions are discussed.
Assuntos
Éter , S-Adenosilmetionina , S-Adenosilmetionina/química , Oxigenases , Éteres , Peptídeos/química , Antibacterianos , Oxigênio , ÁguaRESUMO
[FeFe]-hydrogenases use a unique organometallic complex, termed the H cluster, to reversibly convert H2 into protons and low-potential electrons. It can be best described as a [Fe4S4] cluster coupled to a unique [2Fe]H center where the reaction actually takes place. The latter corresponds to two iron atoms, each of which is bound by one CN- ligand and one CO ligand. The two iron atoms are connected by a unique azadithiolate molecule (-S-CH2-NH-CH2-S-) and an additional bridging CO. This [2Fe]H center is built stepwise thanks to the well-orchestrated action of maturating enzymes that belong to the Hyd machinery. Among them, HydG converts l-tyrosine into CO and CN- to produce a unique l-cysteine-Fe(CO)2CN species termed complex-B. Very recently, HydE was shown to perform radical-based chemistry using synthetic complex-B as a substrate. Here we report the high-resolution crystal structure that establishes the identity of the complex-B-bound HydE. By triggering the reaction prior to crystallization, we trapped a new five-coordinate Fe species, supporting the proposal that HydE performs complex modifications of complex-B to produce a monomeric "SFe(CO)2CN" precursor to the [2Fe]H center. Substrate access, product release, and intermediate transfer are also discussed.
Assuntos
Hidrogenase/química , Proteínas Ferro-Enxofre/química , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.
Assuntos
Biologia Computacional/métodos , Enzimas/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Produtos Biológicos/química , Produtos Biológicos/classificação , Produtos Biológicos/metabolismo , Enzimas/química , Hidroxilação , Metilação , Peptídeos/classificação , Peptídeos/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/fisiologia , Ribossomos/metabolismoRESUMO
Nitrogenase is a key player in the global nitrogen cycle, as it catalyzes the reduction of dinitrogen into ammonia. The active site of the nitrogenase MoFe protein corresponds to a [MoFe7S9C-(R)-homocitrate] species designated FeMo-cofactor, whose biosynthesis and insertion requires the action of over a dozen maturation proteins provided by the NIF (for NItrogen Fixation) assembly machinery. Among them, the radical SAM protein NifB plays an essential role, concomitantly inserting a carbide ion and coupling two [Fe4S4] clusters to form a [Fe8S9C] precursor called NifB-co. Here we report on the X-ray structure of NifB from Methanotrix thermoacetophila at 1.95 Å resolution in a state pending the binding of one [Fe4S4] cluster substrate. The overall NifB architecture indicates that this enzyme has a single SAM binding site, which at this stage is occupied by cysteine residue 62. The structure reveals a unique ligand binding mode for the K1-cluster involving cysteine residues 29 and 128 in addition to histidine 42 and glutamate 65. The latter, together with cysteine 62, belongs to a loop inserted in the active site, likely protecting the already present [Fe4S4] clusters. These two residues regulate the sequence of events, controlling SAM dual reactivity and preventing unwanted radical-based chemistry before the K2 [Fe4S4] cluster substrate is loaded into the protein. The location of the K1-cluster, too far away from the SAM binding site, supports a mechanism in which the K2-cluster is the site of methylation.
Assuntos
Proteínas Arqueais/química , Oxirredutases/química , Proteínas Arqueais/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Cisteína/química , Ácido Glutâmico/química , Histidina/química , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Methanosarcinales/enzimologia , Modelos Químicos , Oxirredutases/metabolismo , Ligação Proteica , Conformação Proteica , S-Adenosilmetionina/metabolismoRESUMO
The synthesis and assembly of the active site [FeFe] unit of [FeFe]-hydrogenases require at least three maturases. The radical S-adenosyl-l-methionine HydG, the best characterized of these proteins, is responsible for the synthesis of the hydrogenase CO and CN(-) ligands from tyrosine-derived dehydroglycine (DHG). We speculated that CN(-) and the CO precursor (-):CO2H may be generated through an elimination reaction. We tested this hypothesis with both wild type and HydG variants defective in second iron-sulfur cluster coordination by measuring the in vitro production of CO, CN(-), and (-):CO2H-derived formate. We indeed observed formate production under these conditions. We conclude that HydG is a multifunctional enzyme that produces DHG, CN(-), and CO at three well-differentiated catalytic sites. We also speculate that homocysteine, cysteine, or a related ligand could be involved in Fe(CO)x(CN)y transfer to the HydF carrier/scaffold.
Assuntos
Monóxido de Carbono/síntese química , Cianetos/síntese química , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Catálise , Domínio Catalítico , Cisteína/química , Desulfovibrio desulfuricans/enzimologia , Homocisteína/química , Hidrogenase/genética , Proteínas Ferro-Enxofre/genética , Ligantes , Estrutura Secundária de Proteína , S-Adenosilmetionina/química , Tirosina/químicaRESUMO
The radical S-adenosyl-l-methionine tryptophan lyase uses radical-based chemistry to convert l-tryptophan into 3-methyl-2-indolic acid, a fragment in the biosynthesis of the thiopeptide antibiotic nosiheptide. This complex reaction involves several successive steps corresponding to (i) the activation by a specific hydrogen-atom abstraction, (ii) an unprecedented â¢CO2- radical migration, (iii) a cyanide fragment release, and (iv) the termination of the radical-based reaction. In vitro study of this reaction is made more difficult because the enzyme produces a significant amount of a shunt product instead of the natural product. Here, using a combination of X-ray crystallography, electron paramagnetic resonance spectroscopy, and quantum and hybrid quantum mechanical/molecular mechanical calculations, we have deciphered the fine mechanism of the key â¢CO2- radical migration, highlighting how the preorganized active site of the protein tightly controls this reaction.
Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Carbono Liases/metabolismo , Triptofano/metabolismo , Proteínas de Bactérias/química , Carbono-Carbono Liases/química , Domínio Catalítico , Cristalografia por Raios X , Descarboxilação , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Modelos Moleculares , Ligação Proteica , Teoria Quântica , Streptomyces/enzimologia , Triptofano/químicaRESUMO
Regiospecific dehydration of vicinal diols by enzymes is a difficult reaction that usually requires activation by dedicated organic cofactors. The enzymatic use of radical-based chemistry is an effective but challenging alternative as radical intermediates are difficult to control. Here we report the X-ray structure of the radical S-adenosyl-l-methionine (SAM) dehydratase AprD4 involved in the biosynthesis of the aminoglycoside (AG) antibiotic apramycin. Using in vitro characterizations and theoretical calculations based on our crystal structure, we have been able to propose a detailed mechanism of AprD4 catalysis, which involves a complex partially substrate-induced proton relay network in the enzyme active site and highlights the key role of the protein matrix in driving high-energy intermediates.
Assuntos
Álcoois/metabolismo , Hidroliases/metabolismo , Prótons , S-Adenosilmetionina/metabolismo , Álcoois/química , Biocatálise , Cristalografia por Raios X , Desidratação , Radicais Livres/química , Radicais Livres/metabolismo , Hidroliases/química , Modelos Moleculares , Teoria Quântica , S-Adenosilmetionina/química , Streptomyces/enzimologia , Especificidade por SubstratoRESUMO
Iron sulfur ([Fe-S]) clusters are essential prosthetic groups involved in fundamental cell processes such as gene expression regulation, electron transfer and Lewis acid base chemistry. Central components of their biogenesis are pyridoxal-5'-phosphate (PLP) dependent l-cysteine desulfurases, which provide the necessary S atoms for [Fe-S] cluster assembly. The archaeon Archaeoglobus fulgidus (Af) has two ORFs, which although annotated as l-cysteine desulfurases of the ISC type (IscS), lack the essential Lys residue (K199 in Af) that forms a Schiff base with PLP. We have previously determined the structure of an Af(IscU-D35A-IscS)2 complex heterologously expressed in Escherichia coli and found it to contain a [Fe2S2] cluster. In order to understand the origin of sulfide in that structure we have performed a series of functional tests using wild type and mutated forms of AfIscS. In addition, we have determined the crystal structure of an AfIscS-D199K mutant. From these studies we conclude that: i) AfIscS has no desulfurase activity; ii) in our in vitro [Fe2S2] cluster assembly experiments, sulfide ions are non-enzymatically generated by a mixture of iron, l-cysteine and PLP and iii) the physiological role of AfIscS may be to provide a cysteine ligand to the nascent cluster as observed in the [Fe2S2]-Af(IscU-D35A-IscS)2 complex. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.
Assuntos
Proteínas Arqueais/metabolismo , Archaeoglobus fulgidus/enzimologia , Liases de Carbono-Enxofre/metabolismo , Cisteína/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Archaeoglobus fulgidus/genética , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/genética , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Ferro/química , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Ligação Proteica , Estrutura Terciária de Proteína , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Homologia de Sequência de Aminoácidos , Sulfetos/química , Sulfetos/metabolismoRESUMO
Fe4S4 clusters are very common versatile prosthetic groups in proteins. Their redox property of being sensitive to O2-induced oxidative damage is, for instance, used by the cell to sense oxygen levels and switch between aerobic and anaerobic metabolisms, as exemplified by the fumarate, nitrate reduction regulator (FNR). Using the hydrogenase maturase HydE from Thermotoga maritima as a template, we obtained several unusual forms of FeS clusters, some of which are associated with important structural changes. These structures represent intermediate states relevant to both FeS cluster assembly and degradation. We observe one Fe2S2 cluster bound by two cysteine persulfide residues. This observation lends structural support to a very recent Raman study, which reported that Fe4S4-to-Fe2S2 cluster conversion upon oxygen exposure in FNR resulted in concomitant production of cysteine persulfide as cluster ligands. Similar persulfide ligands have been observed in vitro for several other Fe4S4 cluster-containing proteins. We have also monitored FeS cluster conversion directly in our protein crystals. Our structures indicate that the Fe4S4-to-Fe2S2 change requires large structural modifications, which are most likely responsible for the dimer-monomer transition in FNR.
Assuntos
Proteínas Ferro-Enxofre/biossíntese , Proteínas Ferro-Enxofre/química , Biossíntese de Proteínas , Proteólise , Cristalografia por Raios X , Ligação Proteica , Thermotoga maritima/enzimologia , Fatores de TempoRESUMO
NosL is a member of a family of radical S-adenosylmethionine enzymes that catalyze the cleavage of the Cα-Cß bond of aromatic amino acids. In this paper, we describe a set of experiments with substrate analogues and mutants for probing the early steps of the NosL mechanism. We provide biochemical evidence in support of the structural studies showing that the 5'-deoxyadenosyl radical abstracts a hydrogen atom from the amino group of tryptophan. We demonstrate that d-tryptophan is a substrate for NosL but shows relaxed regio control of the first ß-scission reaction. Mutagenesis studies confirm that Arg323 is important for controlling the regiochemistry of the ß-scission reaction and that Ser340 binds the substrate by hydrogen bonding to the indolic N1 atom.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Carbono-Carbono Liases/química , Triptofano/química , Substituição de Aminoácidos , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono-Carbono Liases/genética , Carbono-Carbono Liases/metabolismo , Mutagênese Sítio-Dirigida , Especificidade por Substrato , Triptofano/metabolismoRESUMO
The structure of the radical S-adenosyl-L-methionine (SAM) [FeFe]-hydrogenase maturase HydG involved in CN(-) /CO synthesis is characterized by two internal tunnels connecting its tyrosine-binding pocket with the external medium and the C-terminal Fe4 S4 cluster-containing region. A comparison with a tryptophan-bound NosL structure suggests that substrate binding causes the closing of the first tunnel and, along with mutagenesis studies, that tyrosine binds to HydG with its amino group well positioned for H-abstraction by SAM. In this orientation the dehydroglycine (DHG) fragment caused by tyrosine Cα-Cß bond scission can readily migrate through the second tunnel towards the C-terminal domain where both CN(-) and CO are synthesized. Our HydG structure appears to be in a relaxed state with its C-terminal cluster CysX2 CysX22 Cys motif exposed to solvent. A rotation of this domain coupled to Fe4 S4 cluster assembly would bury its putatively reactive unique Fe ion thereby allowing it to interact with DHG.
Assuntos
Proteínas de Bactérias/química , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Thermoanaerobacterium/enzimologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Conformação Proteica , S-Adenosilmetionina/química , Tirosina/químicaRESUMO
Reactions involving H(2), N(2), CO, CO(2) and CH(4) are likely to have been central to the origin of life. This is indicated by the active-site structures of the enzymes involved, which are often reminiscent of minerals. Through the combined efforts of protein crystallography, various types of spectroscopy, theoretical calculations and model chemistry, it has been possible to put forward plausible mechanisms for gas-based metabolism by extant microorganisms. Although the reactions are based on metal centres, the protein matrix regulates reactivity and substrate and product trafficking through internal pathways, specific ligation and dielectricity.
Assuntos
Enzimas/química , Enzimas/metabolismo , Gases/metabolismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Anaerobiose , Biocatálise , Domínio Catalítico , Relação Estrutura-AtividadeRESUMO
The multifunctional nature of Alzheimer's disease calls for MTDLs (multitarget-directed ligands) to act on different components of the pathology, like the cholinergic dysfunction and amyloid aggregation. Such MTDLs are usually on the basis of cholinesterase inhibitors (e.g. tacrine or huprine) coupled with another active molecule aimed at a different target. To aid in the design of these MTDLs, we report the crystal structures of hAChE (human acetylcholinesterase) in complex with FAS-2 (fasciculin 2) and a hydroxylated derivative of huprine (huprine W), and of hBChE (human butyrylcholinesterase) in complex with tacrine. Huprine W in hAChE and tacrine in hBChE reside in strikingly similar positions highlighting the conservation of key interactions, namely, π-π/cation-π interactions with Trp86 (Trp82), and hydrogen bonding with the main chain carbonyl of the catalytic histidine residue. Huprine W forms additional interactions with hAChE, which explains its superior affinity: the isoquinoline moiety is associated with a group of aromatic residues (Tyr337, Phe338 and Phe295 not present in hBChE) in addition to Trp86; the hydroxyl group is hydrogen bonded to both the catalytic serine residue and residues in the oxyanion hole; and the chlorine substituent is nested in a hydrophobic pocket interacting strongly with Trp439. There is no pocket in hBChE that is able to accommodate the chlorine substituent.
Assuntos
Doença de Alzheimer/enzimologia , Aminoquinolinas/química , Inibidores da Colinesterase/química , Colinesterases/química , Colinesterases/metabolismo , Cristalografia por Raios X/métodos , Tacrina/química , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Aminoquinolinas/farmacologia , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Colinesterases/farmacologia , HumanosRESUMO
Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (V(L)) and variable heavy (V(H)) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.
Assuntos
Anticorpos/química , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Proteínas/química , Algoritmos , Sequência de Aminoácidos , Animais , Anticorpos/genética , Anticorpos/metabolismo , Sítios de Ligação/genética , Células CHO , Calorimetria , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Cricetinae , Cricetulus , Cristalografia por Raios X , Humanos , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , TermodinâmicaRESUMO
Streptomyces actuosus tryptophan lyase (NosL) is a radical SAM enzyme which catalyzes the synthesis of 3-methyl-2-indolic acid, a precursor in the synthesis of the promising antibiotic nosiheptide. The reaction involves cleavage of the tryptophan Cα-Cß bond and recombination of the amino-acid-derived -COOH fragment at the indole ring. Reported herein is the 1.8â Å resolution crystal structure of NosL complexed with its substrate. Unexpectedly, only one of the tryptophan amino hydrogen atoms is optimally placed for Hâ abstraction by the SAM-derived 5'-deoxyadenosyl radical. This orientation, in turn, rules out the previously proposed delocalized indole radical as the species which undergoes Cα-Cß bond cleavage. Instead, stereochemical considerations indicate that the reactive intermediate is a (·)NH tryptophanyl radical. A structure-based amino acid sequence comparison of NosL with the tyrosine lyases ThiH and HydG strongly suggests that an equivalent (·)NH radical operates in the latter enzymes.
Assuntos
Triptofano/química , Modelos QuímicosRESUMO
Since the discovery that, despite the active site complexity, only three gene products suffice to obtain active recombinant [FeFe]-hydrogenase, significant light has been shed on this process. Both the source of the CO and CN(-) ligands to iron and the assembly site of the catalytic subcluster are known, and an apo structure of HydF has been published recently. However, the nature of the substrate(s) for the synthesis of the bridging dithiolate ligand to the subcluster remains to be established. From both spectroscopy and model chemistry, it is predicted that an amine function in this ligand plays a central role in catalysis, acting as a base in the heterolytic cleavage of hydrogen.
Assuntos
Hidrogenase/química , Proteínas Ferro-Enxofre/química , Algoritmos , Bioquímica/métodos , Catálise , Domínio Catalítico , Chlamydomonas/metabolismo , Proteínas de Escherichia coli/metabolismo , Giardia/metabolismo , Hidrogênio/química , Ligantes , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Proteínas Recombinantes/química , Espectrofotometria/métodos , Relação Estrutura-Atividade , Thermotoga maritima/metabolismo , Transativadores/metabolismoRESUMO
Darobactins represent a class of ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotics featuring a rare bicyclic structure. They target the Bam-complex of Gram-negative bacteria and exhibit in vivo activity against drug-resistant pathogens. First isolated from Photorhabdus species, the corresponding biosynthetic gene clusters (BGCs) are widespread among γ-proteobacteria, including the genera Vibrio, Yersinia, and Pseudoalteromonas (P.). While the organization of the BGC core is highly conserved, a small subset of Pseudoalteromonas carries an extended BGC with additional genes. Here, we report the identification of brominated and dehydrated darobactin derivatives from P. luteoviolacea strains. The marine derivatives are active against multidrug-resistant (MDR) Gram-negative bacteria and showed solubility and plasma protein binding ability different from darobactin A, rendering it more active than darobactin A. The halogenation reaction is catalyzed by DarH, a new class of flavin-dependent halogenases with a novel fold.