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1.
Biochemistry ; 61(23): 2643-2647, 2022 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-36326713

RESUMO

The radical S-adenosyl-l-methionine (SAM) enzyme TYW1 catalyzes the condensation of C-2 and C-3 atoms of pyruvate with N-methylguanosine containing tRNAPhe to form 4-demethylwyosine (imG-14) modified tRNAPhe. The fate of C-1 is not known, and either formate or carbon dioxide (CO2) has been proposed. In this study, a coupled assay that transforms either CO2 or formate to oxaloacetate (OAA) was used to determine the fate of C-1. In the presence of [1-13C1]-pyruvate, 13C-enriched OAA was observed in a process that is concomitant with the formation of imG-14, under conditions that preferentially transform CO2 and not formate to OAA. These findings are discussed in the context of the cofactor content of TYW1 and a new role for the auxiliary cluster in catalyzing the oxidative cleavage of C-1-C-2 bond of pyruvate in the catalytic cycle of TYW1.


Assuntos
Proteínas Ferro-Enxofre , S-Adenosilmetionina , Dióxido de Carbono , Catálise , Proteínas Ferro-Enxofre/química , Metionina , Estresse Oxidativo , Ácido Pirúvico/química , RNA de Transferência/metabolismo , RNA de Transferência de Fenilalanina/química , S-Adenosilmetionina/metabolismo , Oxirredutases/metabolismo
2.
Biochemistry ; 60(45): 3347-3361, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34730336

RESUMO

Ribosomally synthesized post-translationally modified peptides (RiPPs) are ubiquitous and represent a structurally diverse class of natural products. The ribosomally encoded precursor polypeptides are often extensively modified post-translationally by enzymes that are encoded by coclustered genes. Radical S-adenosyl-l-methionine (SAM) enzymes catalyze numerous chemically challenging transformations. In RiPP biosynthetic pathways, these transformations include the formation of C-H, C-C, C-S, and C-O linkages. In this paper, we show that the Geobacter lovleyi sbtM gene encodes a radical SAM protein, SbtM, which catalyzes the cyclization of a Cys/SeCys residue in a minimal peptide substrate. Biochemical studies of this transformation support a mechanism involving H-atom abstraction at the C-3 of the substrate Cys to initiate the chemistry. Several possible cyclization products were considered. The collective biochemical, spectroscopic, mass spectral, and computational observations point to a thiooxazole as the product of the SbtM-catalyzed modification. To our knowledge, this is the first example of a radical SAM enzyme that catalyzes a transformation involving a SeCys-containing peptide and represents a new paradigm for formation of oxazole-containing RiPP natural products.


Assuntos
Peptídeos Antimicrobianos/metabolismo , Geobacter/metabolismo , S-Adenosilmetionina/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/fisiologia , Peptídeos Antimicrobianos/fisiologia , Produtos Biológicos/metabolismo , Catálise , Geobacter/patogenicidade , Espectrometria de Massas/métodos , Oxazóis , Processamento de Proteína Pós-Traducional/fisiologia , Proteômica/métodos , Ribossomos , S-Adenosilmetionina/metabolismo
3.
Biochemistry ; 60(27): 2179-2185, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34184886

RESUMO

TYW1 is a radical S-adenosyl-l-methionine (SAM) enzyme that catalyzes the condensation of pyruvate and N-methylguanosine-containing tRNAPhe, forming 4-demethylwyosine-containing tRNAPhe. Homologues of TYW1 are found in both archaea and eukarya; archaeal homologues consist of a single domain, while eukaryal homologues contain a flavin binding domain in addition to the radical SAM domain shared with archaeal homologues. In this study, TYW1 from Saccharomyces cerevisiae (ScTYW1) was heterologously expressed in Escherichia coli and purified to homogeneity. ScTYW1 is purified with 0.54 ± 0.07 and 4.2 ± 1.9 equiv of flavin mononucleotide (FMN) and iron, respectively, per mole of protein, suggesting the protein is ∼50% replete with Fe-S clusters and FMN. While both NADPH and NADH are sufficient for activity, significantly more product is observed when used in combination with flavin nucleotides. ScTYW1 is the first example of a radical SAM flavoenzyme that is active with NAD(P)H alone.


Assuntos
Oxirredutases/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Mononucleotídeo de Flavina/metabolismo , NADP/metabolismo
5.
J Am Chem Soc ; 140(22): 6842-6852, 2018 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-29792696

RESUMO

TYW1 is a radical S-adenosyl-l-methionine (SAM) enzyme that catalyzes the condensation of pyruvate and N-methylguanosine to form the posttranscriptional modification, 4-demethylwyosine, in situ on transfer RNA (tRNA). Two mechanisms have been proposed for this transformation, with one of the possible mechanisms invoking a Schiff base intermediate formed between a conserved lysine residue and pyruvate. Utilizing a combination of mass spectrometry and X-ray crystallography, we have obtained evidence to support the formation of a Schiff base lysine adduct in TYW1. When 13C labeled pyruvate is used, the mass shift of the adduct matches that of the labeled pyruvate, indicating that pyruvate is the source of the adduct. Furthermore, a crystal structure of TYW1 provides visualization of the Schiff base lysine-pyruvate adduct, which is positioned directly adjacent to the auxiliary [4Fe-4S] cluster. The adduct coordinates the unique iron of the auxiliary cluster through the lysine nitrogen and a carboxylate oxygen, reminiscent of how the radical SAM [4Fe-4S] cluster is coordinated by SAM. The structure provides insight into the binding site for tRNA and further suggests how radical SAM chemistry can be combined with Schiff base chemistry for RNA modification.


Assuntos
Carboxiliases/metabolismo , Methanocaldococcus/enzimologia , S-Adenosilmetionina/metabolismo , Bases de Schiff/química , Radicais Livres/química , Guanosina/análogos & derivados , Estrutura Molecular , S-Adenosilmetionina/química
6.
Biochemistry ; 56(30): 3874-3876, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28708394

RESUMO

Viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) is a widely distributed protein that is expressed in response to infection and causes antiviral effects against a broad spectrum of viruses. Viperin is a member of the radical S-adenosyl-l-methionine (SAM) superfamily of enzymes, which typically employ a 4Fe-4S cluster to reductively cleave SAM to initiate chemistry. Though the specific reaction catalyzed by viperin remains unknown, it has been shown that expression of viperin causes an increase in the fluidity of lipid membranes, which impedes the budding of nascent viral particles from the membrane inhibiting propagation of the infection. Herein, we show that expression of the human viperin homologue induces a dramatically elongated morphology of the host Escherichia coli cells. Mutation of an essential cysteine that coordinates the radical SAM cluster abrogates this effect. Thus, the native radical SAM activity of viperin is likely occurring in the host bacteria, indicating the elusive substrate is shared between both bacteria and humans, significantly narrowing the range of potential candidate substrates and providing a convenient bacterial platform from which future studies can occur.


Assuntos
Escherichia coli/fisiologia , Proteínas/fisiologia , S-Adenosilmetionina/metabolismo , Substituição de Aminoácidos , Aderência Bacteriana , Cisteína/química , Escherichia coli/citologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Cinética , Microscopia Confocal , Mutagênese Sítio-Dirigida , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
7.
Biochemistry ; 54(23): 3569-72, 2015 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-26052987

RESUMO

TYW1 catalyzes the formation of 4-demethylwyosine via the condensation of N-methylguanosine (m¹G) with carbons 2 and 3 of pyruvate. In this study, labeled transfer ribonucleic acid (tRNA) and pyruvate were utilized to determine the site of hydrogen atom abstraction and regiochemistry of the pyruvate addition. tRNA containing a ²H-labeled m¹G methyl group was used to identify the methyl group of m¹G as the site of hydrogen atom abstraction by 5'-deoxyadenosyl radical. [2-¹³C1-3,3,3-²H3]Pyruvate was used to demonstrate retention of all the pyruvate protons, indicating that C2 of pyruvate forms the bridging carbon of the imidazoline ring and C3 the methyl.


Assuntos
Proteínas Arqueais/metabolismo , Biocatálise , Carboxiliases/metabolismo , Guanosina/análogos & derivados , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , S-Adenosilmetionina/metabolismo , Proteínas Arqueais/química , Radioisótopos de Carbono , Carboxiliases/química , Domínio Catalítico , Deutério , Radicais Livres/química , Radicais Livres/metabolismo , Guanosina/química , Guanosina/metabolismo , Proteínas Ferro-Enxofre/química , Mathanococcus/enzimologia , Metilação , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , RNA Arqueal/química , RNA Arqueal/metabolismo , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/metabolismo , S-Adenosilmetionina/química , Estereoisomerismo
8.
Biochemistry ; 54(18): 2903-10, 2015 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-25933252

RESUMO

The radical S-adenosyl-L-methionine (SAM) superfamily is a large and growing group of enzymes that conduct complex radical-mediated transformations. A one-electron reduction of SAM via the +1 state of the cubane [4Fe-4S] cluster generates a 5'-deoxyadenosyl radical, which initiates turnover. The [4Fe-4S] cluster must be reduced from its resting +2 state to the catalytically active +1 oxidation state by an electron. In practice, dithionite or the Escherichia coli flavodoxin (EcFldA)/ferredoxin (flavodoxin):NADP(+) oxidoreductase (Fpr)/NADPH system is used. Herein, we present a systematic investigation of the reductive activation of the radical SAM enzyme CDG synthase (BsQueE) from Bacillus subtilis comparing biological and chemical reductants. These data show that either of the flavodoxin homologues encoded by the B. subtilis genome, BsYkuN or BsYkuP, as well as a series of small molecule redox mediators, supports BsQueE activity. With dithionite as a reductant, the activity of BsQueE is ~75-fold greater in the presence of BsYkuN and BsYkuP compared to that in the presence of dithionite alone. By contrast, EcFldA supports turnover to ~10-fold greater levels than dithionite alone under the same conditions. Comparing the ratio of the rate of turnover to the apparent binding constant for the flavodoxin homologues reveals 10- and 240-fold preferences for BsYkuN over BsYkuP and EcFldA, respectively. The differential activation of the enzyme cannot be explained by the abortive cleavage of SAM. We conclude from these observations that the differential activation of BsQueE by Fld homologues may reside in the details of the interaction between the flavodoxin and the radical SAM enzyme.


Assuntos
Bacillus subtilis/enzimologia , Radicais Livres/química , S-Adenosilmetionina/química , Ativação Enzimática , Flavodoxina/química , Oxirredução
9.
Nat Chem Biol ; 10(2): 106-12, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24362703

RESUMO

7-carboxy-7-deazaguanine synthase (QueE) catalyzes a key S-adenosyl-L-methionine (AdoMet)- and Mg(2+)-dependent radical-mediated ring contraction step, which is common to the biosynthetic pathways of all deazapurine-containing compounds. QueE is a member of the AdoMet radical superfamily, which employs the 5'-deoxyadenosyl radical from reductive cleavage of AdoMet to initiate chemistry. To provide a mechanistic rationale for this elaborate transformation, we present the crystal structure of a QueE along with structures of pre- and post-turnover states. We find that substrate binds perpendicular to the [4Fe-4S]-bound AdoMet, exposing its C6 hydrogen atom for abstraction and generating the binding site for Mg(2+), which coordinates directly to the substrate. The Burkholderia multivorans structure reported here varies from all other previously characterized members of the AdoMet radical superfamily in that it contains a hypermodified (ß6/α3) protein core and an expanded cluster-binding motif, CX14CX2C.


Assuntos
Magnésio/química , Manganês/química , Modelos Moleculares , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Sódio/química , Sítios de Ligação , Burkholderia/enzimologia , Radicais Livres/química , Radicais Livres/metabolismo , Ligação de Hidrogênio/efeitos dos fármacos , Magnésio/farmacologia , Manganês/farmacologia , Estrutura Molecular , Estrutura Terciária de Proteína , Sódio/farmacologia
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