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1.
Science ; 373(6557): 871-876, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34282049

RESUMO

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.


Assuntos
Aprendizado Profundo , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Proteínas ADAM/química , Sequência de Aminoácidos , Simulação por Computador , Microscopia Crioeletrônica , Cristalografia por Raios X , Bases de Dados de Proteínas , Proteínas de Membrana/química , Modelos Moleculares , Complexos Multiproteicos/química , Redes Neurais de Computação , Subunidades Proteicas/química , Proteínas/fisiologia , Receptores Acoplados a Proteínas G/química , Esfingosina N-Aciltransferase/química
2.
Chembiochem ; 21(5): 663-671, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31512343

RESUMO

We recently reported the discovery of phenylacetate decarboxylase (PhdB), representing one of only ten glycyl-radical-enzyme reaction types known, and a promising biotechnological tool for first-time biochemical synthesis of toluene from renewable resources. Here, we used experimental and computational data to evaluate the plausibility of three candidate PhdB mechanisms, involving either attack at the phenylacetate methylene carbon or carboxyl group [via H-atom abstraction from COOH or single-electron oxidation of COO- (Kolbe-type decarboxylation)]. In vitro experimental data included assays with F-labeled phenylacetate, kinetic studies, and tests with site-directed PhdB mutants; computational data involved estimation of reaction energetics using density functional theory (DFT). The DFT results indicated that all three mechanisms are thermodynamically challenging (beyond the range of many known enzymes in terms of endergonicity or activation energy barrier), reflecting the formidable demands on PhdB for catalysis of this reaction. Evidence that PhdB was able to bind α,α-difluorophenylacetate but was unable to catalyze its decarboxylation supported the enzyme's abstraction of a methylene H atom. Diminished activity of H327A and Y691F mutants was consistent with proposed proton donor roles for His327 and Tyr691. Collectively, these and other data most strongly support PhdB attack at the methylene carbon.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias , Carboxiliases , Tolueno/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carboxiliases/química , Carboxiliases/metabolismo , Cinética , Fenilacetatos , Termodinâmica
3.
Metallomics ; 11(11): 1820-1835, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31532427

RESUMO

Mitochondrial Fe-S cluster biosynthesis is accomplished within yeast utilizing the biophysical attributes of the "Isu1" scaffold assembly protein. As a member of a highly homologous protein family, Isu1 has sequence conservation between orthologs and a conserved ability to assemble [2Fe-2S] clusters. Regardless of species, scaffold orthologs have been shown to exist in both "disordered" and "structured" conformations, a structural architecture that is directly related to conformations utilized during Fe-S cluster assembly. During assembly, the scaffold helps direct the delivery and utilization of Fe(ii) and persulfide substrates to produce [2Fe-2S] clusters, however Zn(ii) binding alters the activity of the scaffold while at the same time stabilizes the protein in its structured state. Additional studies confirm Zn binds to the scaffold's Cys rich active site, and has an impact on the protein's ability to make Fe-S clusters. Understanding the interplay between Fe(ii) and Zn(ii) binding to Isu1 in vitro may help clarify metal loading events that occur during Fe-S cluster assembly in vivo. Here we determine the metal : protein stoichiometry for Isu1 Zn and Fe binding to be 1 : 1 and 2 : 1, respectively. As expected, while Zn binding shifts the Isu1 to its structured state, folding is not influenced by Fe(ii) binding. X-ray absorption spectroscopy (XAS) confirms Zn(ii) binds to the scaffold's cysteine rich active site but Fe(ii) binds at a location distinct from the active site. XAS results show Isu1 binding initially of either Fe(ii) or Zn(ii) does not significantly perturb the metal site structure of alternate metal. XAS confirmed that four scaffold orthologs bind iron as high-spin Fe(ii) at a site composed of ca. 6 oxygen and nitrogen nearest neighbor ligands. Finally, in our report Zn binding dramatically reduces the Fe-S cluster assembly activity of Isu1 even in the presence of frataxin. Given the Fe-binding activity we report for Isu1 and its orthologs here, a possible mechanism involving Fe(ii) transport to the scaffold's active site during cluster assembly has been considered.


Assuntos
Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Zinco/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Proteínas Mitocondriais/química , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Espectroscopia por Absorção de Raios X
4.
Biomol NMR Assign ; 13(2): 377-381, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31440902

RESUMO

Friedreich's ataxia, the most prevalent hereditary ataxia, is caused by a patient's inability to produce a viable form of the protein frataxin. Frataxin plays an essential role in cellular iron regulation and has been shown to participate in the assembly of iron-sulfur (Fe-S) clusters under a variety of roles, including modulating persulfide production and directing Fe(II) delivery to the assembly scaffold protein. While the activity and structure of multiple eukaryotic frataxin orthologs have been characterized, the fly ortholog has numerous advantages over other orthologs with regards to protein stability, its activity towards Fe-S cluster assembly and its stability for forming stable proteins partner assemblies. Given the obvious advantages for studying the Drosophila melanogaster frataxin homolog (Dfh) over its orthologs, we have undertaken a structural characterization of apo-Dfh as the first step towards solving the solution structure of the protein alone and in complex with protein partners within the Fe-S cluster assembly pathway.


Assuntos
Apoproteínas/química , Drosophila melanogaster , Proteínas de Ligação ao Ferro/química , Ressonância Magnética Nuclear Biomolecular , Animais , Estrutura Secundária de Proteína
5.
Nat Chem Biol ; 14(5): 451-457, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29556105

RESUMO

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum.


Assuntos
Bactérias/enzimologia , Microbiota , Tolueno/metabolismo , Acidobacteria/enzimologia , Acidobacteria/genética , Acidobacteria/isolamento & purificação , Anaerobiose , Bactérias/genética , Biomassa , Carboxiliases/metabolismo , Catálise , Genes Bacterianos , Sedimentos Geológicos/microbiologia , Lagos/microbiologia , Lignina/química , Funções Verossimilhança , Metagenômica , Fenilacetatos/química , Filogenia , Proteômica , Proteínas Recombinantes/metabolismo , Esgotos/microbiologia
6.
Biotechnol Biofuels ; 11: 340, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30607175

RESUMO

Background: Geranylgeranyl reductase (GGR) is a flavin-containing redox enzyme that hydrogenates a variety of unactivated polyprenyl substrates, which are further processed mostly for lipid biosynthesis in archaea or chlorophyll biosynthesis in plants. To date, only a few GGR genes have been confirmed to reduce polyprenyl substrates in vitro or in vivo. Results: In this work, we aimed to expand the confirmed GGR activity space by searching for novel genes that function under amenable conditions for microbial mesophilic growth in conventional hosts such as Escherichia coli or Saccharomyces cerevisiae. 31 putative GGRs were selected to test for potential reductase activity in vitro on farnesyl pyrophosphate, geranylgeranyl pyrophosphate, farnesol (FOH), and geranylgeraniol (GGOH). We report the discovery of several novel GGRs exhibiting significant activity toward various polyprenyl substrates under mild conditions (i.e., pH 7.4, T = 37 °C), including the discovery of a novel bacterial GGR isolated from Streptomyces coelicolor. In addition, we uncover new mechanistic insights within several GGR variants, including GGR-mediated phosphatase activity toward polyprenyl pyrophosphates and the first demonstration of completely hydrogenated GGOH and FOH substrates. Conclusion: These collective results enhance the potential for metabolic engineers to manufacture a variety of isoprenoid-based biofuels, polymers, and chemical feedstocks in common microbial hosts such as E. coli or S. cerevisiae.

7.
J Am Chem Soc ; 138(38): 12459-71, 2016 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-27562882

RESUMO

Metal-nitroxyl (M-HNO/M-NO(-)) coordination units are found in denitrification enzymes of the global nitrogen cycle, and free HNO exhibits pharmacological properties related to cardiovascular physiology that are distinct from nitric oxide (NO). To elucidate the properties that control the binding and release of coordinated nitroxyl or its anion at these biological metal sites, we synthesized {CoNO}(8) (1, 2) and {CoNO}(9) (3, 4) complexes that contain diimine-dipyrrolide supporting ligands. Experimental (NMR, IR, MS, EPR, XAS, XRD) and computational data (DFT) support an oxidation state assignment for 3 and 4 of high spin Co(II) (SCo = 3/2) coordinated to (3)NO(-) (SNO = 1) for Stot = 1/2. As suggested by DFT, upon protonation, a spin transition occurs to generate a putative low spin Co(II)-(1)HNO (SCo = Stot = 1/2); the Co-NO bond is ∼0.2 Šlonger, more labile, and facilitates the release of HNO. This property was confirmed experimentally through the detection and quantification of N2O (∼70% yield), a byproduct of the established HNO self-reaction (2HNO → N2O + H2O). Additionally, 3 and 4 function as HNO donors in aqueous media at pH 7.4 and react with known HNO targets, such as a water-soluble Mn(III)-porphyrin ([Mn(III)(TPPS)](3-); TPPS = meso-tetrakis(4-sulfonatophenyl)porphyrinate) and ferric myoglobin (metMb) to quantitatively yield [Mn(TPPS)(NO)](4-) and MbNO, respectively.


Assuntos
Metaloproteínas/química , Metaloproteínas/metabolismo , Óxidos de Nitrogênio/química , Eletroquímica , Modelos Moleculares , Estrutura Molecular , Ciclo do Nitrogênio
8.
Proc Natl Acad Sci U S A ; 113(17): 4700-5, 2016 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-27071088

RESUMO

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a key protein involved in pre-mRNA processing. CPSF30 contains five Cys3His domains (annotated as "zinc-finger" domains). Using inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy, and UV-visible spectroscopy, we report that CPSF30 is isolated with iron, in addition to zinc. Iron is present in CPSF30 as a 2Fe-2S cluster and uses one of the Cys3His domains; 2Fe-2S clusters with a Cys3His ligand set are rare and notably have also been identified in MitoNEET, a protein that was also annotated as a zinc finger. These findings support a role for iron in some zinc-finger proteins. Using electrophoretic mobility shift assays and fluorescence anisotropy, we report that CPSF30 selectively recognizes the AU-rich hexamer (AAUAAA) sequence present in pre-mRNA, providing the first molecular-based evidence to our knowledge for CPSF30/RNA binding. Removal of zinc, or both zinc and iron, abrogates binding, whereas removal of just iron significantly lessens binding. From these data we propose a model for RNA recognition that involves a metal-dependent cooperative binding mechanism.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/química , Ferro/química , Sinais de Poliadenilação na Ponta 3' do RNA/genética , RNA Mensageiro/química , Enxofre/química , Fatores de Poliadenilação e Clivagem de mRNA/química , Sítios de Ligação , Fator de Especificidade de Clivagem e Poliadenilação/genética , Simulação por Computador , Humanos , Modelos Químicos , Poliadenilação/genética , Ligação Proteica , Fatores de Poliadenilação e Clivagem de mRNA/genética
9.
Biometals ; 28(3): 567-76, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25782577

RESUMO

Iron-sulfur (Fe-S) cluster containing proteins are utilized in almost every biochemical pathway. The unique redox and coordination chemistry associated with the cofactor allows these proteins to participate in a diverse set of reactions, including electron transfer, enzyme catalysis, DNA synthesis and signaling within several pathways. Due to the high reactivity of the metal, it is not surprising that biological Fe-S cluster assembly is tightly regulated within cells. In yeast, the major assembly pathway for Fe-S clusters is the mitochondrial ISC pathway. Yeast Fe-S cluster assembly is accomplished using the scaffold protein (Isu1) as the molecular foundation, with assistance from the cysteine desulfurase (Nfs1) to provide sulfur, the accessory protein (Isd11) to regulate Nfs1 activity, the yeast frataxin homologue (Yfh1) to regulate Nfs1 activity and participate in Isu1 Fe loading possibly as a chaperone, and the ferredoxin (Yah1) to provide reducing equivalents for assembly. In this report, we utilize calorimetric and spectroscopic methods to provide molecular insight into how wt-Isu1 from S. cerevisiae becomes loaded with iron. Isothermal titration calorimetry and an iron competition binding assay were developed to characterize the energetics of protein Fe(II) binding. Differential scanning calorimetry was used to identify thermodynamic characteristics of the protein in the apo state or under iron loaded conditions. Finally, X-ray absorption spectroscopy was used to characterize the electronic and structural properties of Fe(II) bound to Isu1. Current data are compared to our previous characterization of the D37A Isu1 mutant, and these suggest that when Isu1 binds Fe(II) in a manner not perturbed by the D37A substitution, and that metal binding occurs at a site distinct from the cysteine rich active site in the protein.


Assuntos
Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Domínio Catalítico , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
J Am Chem Soc ; 136(36): 12560-3, 2014 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-25073017

RESUMO

Research on the one-electron reduced analogue of NO, namely nitroxyl (HNO/NO(-)), has revealed distinguishing properties regarding its utility as a therapeutic. However, the fleeting nature of HNO requires the design of donor molecules. Metal nitrosyl (MNO) complexes could serve as potential HNO donors. The synthesis, spectroscopic/structural characterization, and HNO donor properties of a {CoNO}(8) complex in a pyrrole/imine ligand frame are reported. The {CoNO}(8) complex [Co(LN4(PhCl))(NO)] (1) does not react with established HNO targets such as Fe(III) hemes or Ph3P. However, in the presence of stoichiometric H(+) 1 behaves as an HNO donor. Complex 1 readily reacts with [Fe(TPP)Cl] or Ph3P to afford the {FeNO}(7) porphyrin or Ph3P═O/Ph3P═NH, respectively. In the absence of an HNO target, the {Co(NO)2}(10) dinitrosyl (3) is the end product. Complex 1 also reacts with O2 to yield the corresponding Co(III)-η(1)-ONO2 (2) nitrato analogue. This report is the first to suggest an HNO donor role for {CoNO}(8) with biotargets such as Fe(III)-porphyrins.


Assuntos
Cobalto/química , Óxido Nítrico/química , Óxidos de Nitrogênio/química , Compostos Organometálicos/química , Prótons , Modelos Moleculares , Estrutura Molecular
11.
Curr Opin Chem Biol ; 15(2): 312-8, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21288761

RESUMO

Protein controlled iron homeostasis is essential for maintaining appropriate levels and availability of metal within cells. Recently, two iron chaperones have been discovered that direct metal within two unique pathways: (1) mitochondrial iron-sulfur (Fe-S) cluster assembly and (2) within the ferritin iron storage system. Although structural and functional details describing how these iron chaperones operate are emerging, both share similar iron binding affinities and metal-ligand site structures that enable them to bind and release Fe2+ to specific protein partners. Molecular details related to iron binding and delivery by these chaperones will be explored within this review.


Assuntos
Ferritinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Humanos , Modelos Moleculares
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