RESUMO
Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and membrane-associated nature of the respiratory reductive dehalogenases has hindered their detailed kinetic study. In contrast, the evolutionarily related catabolic reductive dehalogenases are oxygen tolerant, with those that are naturally fused to a reductase domain with similarity to phthalate dioxygenase presenting attractive targets for further study. We present efficient heterologous expression of a self-sufficient catabolic reductive dehalogenase from Jhaorihella thermophila in Escherichia coli. Combining the use of maltose-binding protein as a solubility-enhancing tag with the btuCEDFB cobalamin uptake system affords up to 40% cobalamin occupancy and a full complement of iron-sulfur clusters. The enzyme is able to efficiently perform NADPH-dependent dehalogenation of brominated and iodinated phenolic compounds, including the flame retardant tetrabromobisphenol, under both anaerobic and aerobic conditions. NADPH consumption is tightly coupled to product formation. Surprisingly, corresponding chlorinated compounds only act as competitive inhibitors. Electron paramagnetic resonance spectroscopy reveals loss of the Co(II) signal observed in the resting state of the enzyme under steady-state conditions, suggesting accumulation of Co(I)/(III) species prior to the rate-limiting step. In vivo reductive debromination activity is readily observed, and when the enzyme is expressed in E. coli strain W, supports growth on 3-bromo-4-hydroxyphenylacetic as a sole carbon source. This demonstrates the potential for catabolic reductive dehalogenases for future application in bioremediation.
Assuntos
Hidrolases , NADP , Rhodobacteraceae , Escherichia coli/genética , NADP/metabolismo , Oxigênio/química , Vitamina B 12/metabolismo , Fenóis/química , Fenóis/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Hidrolases/química , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/metabolismo , Rhodobacteraceae/enzimologia , Rhodobacteraceae/genética , Estrutura Terciária de Proteína , Modelos Moleculares , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Coenzimas/metabolismoRESUMO
The ubiquitous UbiD family of reversible decarboxylases is implicated in a wide range of microbial processes and depends on the prenylated flavin mononucleotide cofactor for catalysis. However, only a handful of UbiD family members have been characterized in detail, and comparison between these has suggested considerable variability in enzyme dynamics and mechanism linked to substrate specificity. In this study, we provide structural and biochemical insights into the indole-3-carboxylic acid decarboxylase, representing an UbiD enzyme activity distinct from those previously studied. Structural insights from crystal structure determination combined with small-angle X-ray scattering measurements reveal that the enzyme likely undergoes an open-closed transition as a consequence of domain motion, an event that is likely coupled to catalysis. We also demonstrate that the indole-3-carboxylic acid decarboxylase can be coupled with carboxylic acid reductase to produce indole-3-carboxyaldehyde from indole + CO2 under ambient conditions. These insights provide further evidence for a common mode of action in the widespread UbiD enzyme family.
Assuntos
Carboxiliases , Modelos Moleculares , Carboxiliases/química , Carboxiliases/metabolismo , Mononucleotídeo de Flavina/metabolismo , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge. Here we determine the crystal structures of the multi-subunit vanillic acid decarboxylase VdcCD. We demonstrate that the small VdcD subunit acts as an allosteric activator of the UbiD-like VdcC. Comparison of distinct VdcCD structures reveals domain motion of the prFMN-binding domain directly affects active site architecture. Docking of substrate and prFMN-adduct species reveals active site reorganisation coupled to domain motion supports rearrangement of the substrate aromatic moiety. Together with kinetic solvent viscosity effects, this establishes prFMN covalent catalysis of aromatic (de)carboxylation is afforded by UbiD dynamics.
Assuntos
Carboxiliases/química , Carboxiliases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Reação de Cicloadição , Descarboxilação , Mononucleotídeo de Flavina/metabolismo , Cinética , Modelos Moleculares , Oxigênio/farmacologia , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Solventes , Relação Estrutura-Atividade , Especificidade por Substrato , ViscosidadeRESUMO
The UbiD family of reversible (de)carboxylases depends on the recently discovered prenylated-FMN (prFMN) cofactor for activity. The model enzyme ferulic acid decarboxylase (Fdc1) decarboxylates unsaturated aliphatic acids via a reversible 1,3-cycloaddition process. Protein engineering has extended the Fdc1 substrate range to include (hetero)aromatic acids, although catalytic rates remain poor. This raises the question how efficient decarboxylation of (hetero)aromatic acids is achieved by other UbiD family members. Here, we show that the Pseudomonas aeruginosa virulence attenuation factor PA0254/HudA is a pyrrole-2-carboxylic acid decarboxylase. The crystal structure of the enzyme in the presence of the reversible inhibitor imidazole reveals a covalent prFMN-imidazole adduct is formed. Substrate screening reveals HudA and selected active site variants can accept a modest range of heteroaromatic compounds, including thiophene-2-carboxylic acid. Together with computational studies, our data suggests prFMN covalent catalysis occurs via electrophilic aromatic substitution and links HudA activity with the inhibitory effects of pyrrole-2-carboxylic acid on P. aeruginosa quorum sensing.
RESUMO
Conjugated alkenes such as dienes and polyenes have a range of applications as pharmaceutical agents and valuable building blocks in the polymer industry. Development of a renewable route to these compounds provides an alternative to fossil fuel derived production. The enzyme family of the UbiD decarboxylases offers substantial scope for alkene production, readily converting poly unsaturated acids. However, biochemical pathways producing the required substrates are poorly characterized, and UbiD-application has hitherto been limited to biological styrene production. Herein, we present a proof-of-principle study for microbial production of polyenes using a bioinspired strategy employing a polyketide synthase (PKS) in combination with a UbiD-enzyme. Deconstructing a bacterial iterative type II PKS enabled repurposing the broad-spectrum antibiotic andrimid biosynthesis pathway to access the metabolic intermediate 2,4,6-octatrienoic acid, a valuable chemical for material and pharmaceutical industry. Combination with the fungal ferulic acid decarboxylase (Fdc1) led to a biocatalytic cascade-type reaction for the production of hepta-1,3,5-triene in vivo. Our approach provides a novel route to generate unsaturated hydrocarbons and related chemicals and provides a blue-print for future development and application.
Assuntos
Antibacterianos/metabolismo , Aspergillus niger/enzimologia , Carboxiliases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas Fúngicas/metabolismo , Engenharia Metabólica/métodos , Biocatálise , Escherichia coli/genética , Ácidos Graxos Insaturados/metabolismo , Polienos/metabolismo , Policetídeo Sintases/metabolismo , Pirróis/metabolismoRESUMO
The family of cobalamin class-III dependent enzymes is composed of the reductive dehalogenases (RDases) and related epoxyqueuosine reductases. RDases are crucial for the energy conserving process of organohalide respiration. These enzymes have the ability to reductively cleave carbon-halogen bonds, present in a number of environmentally hazardous pollutants, making them of significant interest for bioremediation applications. Unfortunately, it is difficult to obtain sufficient yields of pure RDase isolated from organohalide respiring bacteria for biochemical studies. Hence, robust heterologous expression systems are required that yield the active holo-enzyme which requires both iron-sulphur cluster and cobalamin incorporation. We present a comparative study of the heterologous expression strains Bacillus megaterium, Escherichia coli HMS174(DE3), Shimwellia blattae and a commercial strain of Vibrio natrigenes, for cobalamin class-III dependent enzymes expression. The Nitratireductor pacificus pht-3B reductive dehalogenase (NpRdhA) and the epoxyqueuosine reductase from Streptococcus thermophilus (StoQ) were used as model enzymes. We also analysed whether co-expression of the cobalamin transporter BtuB, supports increased cobalamin incorporation into these enzymes in E. coli. We conclude that while expression in Bacillus megaterium resulted in the highest levels of cofactor incorporation, co-expression of BtuB in E. coli presents an appropriate balance between cofactor incorporation and protein yield in both cases.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Expressão Gênica , Proteínas Ferro-Enxofre/genética , Oxirredutases/genética , Vitamina B 12/química , Bacillus megaterium/enzimologia , Bacillus megaterium/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biodegradação Ambiental , Clonagem Molecular , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Escherichia coli/enzimologia , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Halogênios/química , Halogênios/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Cinética , Modelos Moleculares , Nucleosídeo Q/análogos & derivados , Nucleosídeo Q/química , Nucleosídeo Q/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Phyllobacteriaceae/enzimologia , Phyllobacteriaceae/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus thermophilus/enzimologia , Streptococcus thermophilus/genética , Vibrio/enzimologia , Vibrio/genética , Vitamina B 12/metabolismoRESUMO
Reductive dehalogenases are responsible for the reductive cleavage of carbon-halogen bonds during organohalide respiration. A variety of mechanisms have been proposed for these cobalamin and [4Fe-4S] containing enzymes, including organocobalt, radical, or cobalt-halide adduct based catalysis. The latter was proposed for the oxygen-tolerant Nitratireductor pacificus pht-3B catabolic reductive dehalogenase (NpRdhA). Here, we present the first substrate bound NpRdhA crystal structures, confirming a direct cobalt-halogen interaction is established and providing a rationale for substrate preference. Product formation is observed in crystallo due to X-ray photoreduction. Protein engineering enables rational alteration of substrate preference, providing a future blue print for the application of this and related enzymes in bioremediation.
RESUMO
The UbiD enzyme plays an important role in bacterial ubiquinone (coenzyme Q) biosynthesis. It belongs to a family of reversible decarboxylases that interconvert propenoic or aromatic acids with the corresponding alkenes or aromatic compounds using a prenylated flavin mononucleotide cofactor. This cofactor is suggested to support (de)carboxylation through a reversible 1,3-dipolar cycloaddition process. Here, we report an atomic-level description of the reaction of the UbiD-related ferulic acid decarboxylase with substituted propenoic and propiolic acids (data ranging from 1.01-1.39 Å). The enzyme is only able to couple (de)carboxylation of cinnamic acid-type compounds to reversible 1,3-dipolar cycloaddition, while the formation of dead-end prenylated flavin mononucleotide cycloadducts occurs with distinct propenoic and propiolic acids. The active site imposes considerable strain on covalent intermediates formed with cinnamic and phenylpropiolic acids. Strain reduction through mutagenesis negatively affects catalytic rates with cinnamic acid, indicating a direct link between enzyme-induced strain and catalysis that is supported by computational studies.
Assuntos
Alcinos/metabolismo , Carboxiliases/metabolismo , Propionatos/metabolismo , Alcinos/química , Biocatálise , Carboxiliases/química , Carboxiliases/isolamento & purificação , Reação de Cicloadição , Teoria da Densidade Funcional , Modelos Moleculares , Conformação Molecular , Propionatos/químicaRESUMO
The recent discovery of the prenylated FMN (prFMN) cofactor has led to a renewed interest in the prFMN-dependent UbiD family of enzymes. The latter catalyses the reversible decarboxylation of alpha-beta unsaturated carboxylic acids and features widely in microbial metabolism. The flavin prenyltransferase UbiX synthesizes prFMN from reduced FMN and phosphorylated dimethylallyl precursors. Oxidative maturation of the resulting prFMNreduced species to the active prFMNiminium form is required for UbiD activity. Heterologous production of active holo-UbiD requires co-expression of UbiX, but the levels of prFMN incorporation and oxidative maturation appear variable. Detailed protocols and strategies for in vitro reconstitution and oxidative maturation of UbiD are presented that can yield an alternative source of active holo-UbiD for biochemical studies.
Assuntos
Carboxiliases/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Carboxiliases/isolamento & purificação , Carboxiliases/metabolismo , Descarboxilação , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Mononucleotídeo de Flavina/química , Oxirredução , Prenilação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The UbiX-UbiD enzymes are widespread in microbes, acting in concert to decarboxylate alpha-beta unsaturated carboxylic acids using a highly modified flavin cofactor, prenylated FMN (prFMN). UbiX serves as the flavin prenyltransferase, extending the isoalloxazine ring system with a fourth non-aromatic ring, derived from sequential linkage between a dimethylallyl moiety and the FMN N5 and C6. Using structure determination and solution studies of both dimethylallyl monophosphate (DMAP) and dimethyallyl pyrophosphate (DMAPP) dependent UbiX enzymes, we reveal the first step, N5-C1' bond formation, is contingent on the presence of a dimethylallyl substrate moiety. Hence, an SN1 mechanism similar to other prenyltransferases is proposed. Selected variants of the (pyro)phosphate binding site are unable to catalyse subsequent Friedel-Crafts alkylation of the flavin C6, but can be rescued by addition of (pyro)phosphate. Thus, retention of the (pyro)phosphate leaving group is required for C6-C3' bond formation, resembling pyrophosphate initiated class I terpene cyclase reaction chemistry.
Assuntos
Aspergillus niger/enzimologia , Carboxiliases/metabolismo , Dimetilaliltranstransferase/metabolismo , Dinitrocresóis/metabolismo , Proteínas Fúngicas/metabolismo , Sítios de Ligação , Descarboxilação , Difosfatos/metabolismo , Prenilação , Terpenos/metabolismoRESUMO
The biological production of FDCA is of considerable value as a potential replacement for petrochemical-derived monomers such as terephthalate, used in polyethylene terephthalate (PET) plastics. HmfF belongs to an uncharacterized branch of the prenylated flavin (prFMN) dependent UbiD family of reversible (de)carboxylases and is proposed to convert 2,5-furandicarboxylic acid (FDCA) to furoic acid in vivo. We present a detailed characterization of HmfF and demonstrate that HmfF can catalyze furoic acid carboxylation at elevated CO2 levels in vitro. We report the crystal structure of a thermophilic HmfF from Pelotomaculum thermopropionicum, revealing that the active site located above the prFMN cofactor contains a furoic acid/FDCA binding site composed of residues H296-R304-R331 specific to the HmfF branch of UbiD enzymes. Variants of the latter are compromised in activity, while H296N alters the substrate preference to pyrrole compounds. Solution studies and crystal structure determination of an engineered dimeric form of the enzyme revealed an unexpected key role for a UbiD family wide conserved Leu residue in activity. The structural insights into substrate and cofactor binding provide a template for further exploitation of HmfF in the production of FDCA plastic precursors and improve our understanding of catalysis by members of the UbiD enzyme family.
RESUMO
Fungal ferulic acid decarboxylases (FDCs) belong to the UbiD-family of enzymes and catalyse the reversible (de)carboxylation of cinnamic acid derivatives through the use of a prenylated flavin cofactor. The latter is synthesised by the flavin prenyltransferase UbiX. Herein, we demonstrate the applicability of FDC/UbiX expressing cells for both isolated enzyme and whole-cell biocatalysis. FDCs exhibit high activity with total turnover numbers (TTN) of up to 55000 and turnover frequency (TOF) of up to 370â min-1. Co-solvent compatibility studies revealed FDC's tolerance to some organic solvents up 20 % v/v. Using the in-vitro (de)carboxylase activity of holo-FDC as well as whole-cell biocatalysts, we performed a substrate profiling study of three FDCs, providing insights into structural determinants of activity. FDCs display broad substrate tolerance towards a wide range of acrylic acid derivatives bearing (hetero)cyclic or olefinic substituents at C3 affording conversions of up to >99 %. The synthetic utility of FDCs was demonstrated by a preparative-scale decarboxylation.
RESUMO
Reductive dehalogenases are corrinoid and iron-sulfur cluster-dependent enzymes that mostly act as the terminal oxidoreductases in the bacterial organohalide respiration process. This process often leads to detoxification of recalcitrant organohalide pollutants. While low cell yields and oxygen sensitivity hamper the study of many reductive dehalogenases, this is not the case for the nonrespiratory reductive dehalogenase NpRdhA from Nitratireductor pacificus. We here report in vitro and in vivo reconstitution of an NADPH-dependent reducing system for NpRdhA. Surprisingly, NpRdhA mediated organohalide reduction could not be supported using N. pacificus ferredoxin-NAD(P)H oxidoreductase and associated ferredoxins. Instead, we found a nonphysiological system comprised of the Escherichia coli flavodoxin reductase (EcFldr) in combination with spinach ferredoxin (SpFd) was able to support NADPH-dependent organohalide reduction by NpRdhA. Using this system, organohalide reduction can be performed under both anaerobic and aerobic conditions, with 1.1 ± 0.1 and 3.5 ± 0.3 equiv of NADPH consumed per product produced, respectively. No significant enzyme inactivation under aerobic conditions was observed, suggesting a Co(I) species is unlikely to be present under steady state conditions. Furthermore, reduction of the Co(II) resting state was not observed in the absence of substrate. Only the coexpression of EcFldr, SpFd, and NpRdhA in Bacillus megaterium conferred the latter with the ability to reduce brominated NpRdhA substrates in vivo, in agreement with our in vitro observations. Our work provides new insights into biological reductive dehalogenase reduction and establishes a blueprint for the minimal functional organohalide reduction module required for bioremediation in situ.
Assuntos
Proteínas de Bactérias/metabolismo , NADH NADPH Oxirredutases/metabolismo , NADP/metabolismo , Phyllobacteriaceae/enzimologia , Proteínas de Bactérias/química , Halogenação , Concentração de Íons de Hidrogênio , Modelos Moleculares , NADH NADPH Oxirredutases/química , Concentração Osmolar , Oxirredução , Phyllobacteriaceae/química , Phyllobacteriaceae/metabolismoRESUMO
Reductive dehalogenases (RDases) are key enzymes involved in the respiratory process of anaerobic organohalide respiring bacteria (ORB). Heterologous expression of respiratory RDases is desirable for structural and functional studies; however, there are few reports of successful expression of these enzymes. Dehalobacter sp. strain UNSWDHB is an ORB, whose preferred electron acceptor is chloroform. This study describes efforts to express recombinant reductive dehalogenase (TmrA), derived from UNSW DHB, using the heterologous hosts Escherichia coli and Bacillus megaterium. Here, we report the recombinant expression of soluble and functional TmrA, using B. megaterium as an expression host under a xylose-inducible promoter. Successful incorporation of iron-sulfur clusters and a corrinoid cofactor was demonstrated using UV-vis spectroscopic analyses. In vitro dehalogenation of chloroform using purified recombinant TmrA was demonstrated. This is the first known report of heterologous expression and purification of a respiratory reductive dehalogenase from an obligate organohalide respiring bacterium.
Assuntos
Bacillus megaterium/genética , Clorofórmio/química , Oxirredutases/genética , Halogenação , Proteínas Recombinantes/genéticaRESUMO
The UbiD family of reversible decarboxylases act on aromatic, heteroaromatic, and unsaturated aliphatic acids and utilize a prenylated flavin mononucleotide (prFMN) as cofactor, bound adjacent to a conserved Glu-Arg-Glu/Asp ionic network in the enzyme's active site. It is proposed that UbiD activation requires oxidative maturation of the cofactor, for which two distinct isomers, prFMNketimine and prFMNiminium, have been observed. It also has been suggested that only the prFMNiminium form is relevant to catalysis, which requires transient cycloaddition between substrate and cofactor. Using Aspergillus niger Fdc1 as a model system, we reveal that isomerization of prFMNiminium to prFMNketimine is a light-dependent process that is largely independent of the Glu277-Arg173-Glu282 network and accompanied by irreversible loss of activity. On the other hand, efficient catalysis was highly dependent on an intact Glu-Arg-Glu network, as only Glu â Asp substitutions retain activity. Surprisingly, oxidative maturation to form the prFMNiminium species is severely affected only for the R173A variant. In summary, the unusual irreversible isomerization of prFMN is light-dependent and probably proceeds via high-energy intermediates but is independent of the Glu-Arg-Glu network. Our results from mutagenesis, crystallographic, spectroscopic, and kinetic experiments indicate a clear role for the Glu-Arg-Glu network in both catalysis and oxidative maturation.
Assuntos
Aspergillus niger/enzimologia , Carboxiliases/química , Carboxiliases/metabolismo , Mononucleotídeo de Flavina/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Aspergillus niger/química , Aspergillus niger/genética , Sítios de Ligação , Carboxiliases/genética , Catálise , Domínio Catalítico , Sequência Conservada , Mononucleotídeo de Flavina/química , Proteínas Fúngicas/genética , Isomerismo , Cinética , OxirreduçãoRESUMO
The UbiX-UbiD system consists of the flavin prenyltransferase UbiX that produces prenylated FMN that serves as the cofactor for the (de)carboxylase UbiD. Recent developments have provided structural insights into the mechanism of both enzymes, detailing unusual chemistry in each case. The proposed reversible 1,3-dipolar cycloaddition between the cofactor and substrate serves as a model to explain many of the key UbiD family features. However, considerable variation exists in the many branches of the UbiD family tree.
Assuntos
Carboxiliases , Dimetilaliltranstransferase , Proteínas de Escherichia coli , Escherichia coli , Flavinas , Flavoproteínas , Prenilação/fisiologia , Carboxiliases/química , Carboxiliases/genética , Carboxiliases/metabolismo , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flavinas/biossíntese , Flavinas/química , Flavinas/genética , Flavoproteínas/química , Flavoproteínas/genética , Flavoproteínas/metabolismoRESUMO
The activity of the reversible decarboxylase enzyme Fdc1 is dependent on prenylated FMN (prFMN), a recently discovered cofactor. The oxidized prFMN supports a 1,3-dipolar cycloaddition mechanism that underpins reversible decarboxylation. Fdc1 is a distinct member of the UbiD family of enzymes, with the canonical UbiD catalyzing the (de)carboxylation of para-hydroxybenzoic acid-type substrates. Here we show that the Escherichia coli UbiD enzyme, which is implicated in ubiquinone biosynthesis, cannot be isolated in an active holoenzyme form despite the fact active holoFdc1 is readily obtained. Formation of holoUbiD requires reconstitution in vitro of the apoUbiD with reduced prFMN. Furthermore, although the Fdc1 apoenzyme can be readily reconstituted and activated, in vitro oxidation to the mature prFMN cofactor stalls at formation of a radical prFMN species in holoUbiD. Further oxidative maturation in vitro occurs only at alkaline pH, suggesting a proton-coupled electron transfer precedes formation of the fully oxidized prFMN. Crystal structures of holoUbiD reveal a relatively open active site potentially occluded from solvent through domain motion. The presence of a prFMN sulfite-adduct in one of the UbiD crystal structures confirms oxidative maturation does occur at ambient pH on a slow time scale. Activity could not be detected for a range of putative para-hydroxybenzoic acid substrates tested. However, the lack of an obvious hydrophobic binding pocket for the octaprenyl tail of the proposed ubiquinone precursor substrate does suggest UbiD might act on a non-prenylated precursor. Our data reveals an unexpected variation occurs in domain mobility, prFMN binding, and maturation by the UbiD enzyme family.
Assuntos
Carboxiliases/metabolismo , Escherichia coli/metabolismo , Mononucleotídeo de Flavina/metabolismo , Ubiquinona/metabolismo , Sítios de Ligação , Carboxiliases/química , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Escherichia coli/química , Modelos Moleculares , Oxirredução , Prenilação , Domínios Proteicos , Ubiquinona/químicaRESUMO
Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1(Ubix), the cofactor confers structural stability to the enzyme. IM-MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM-MS data.
Assuntos
Carboxiliases/química , Carboxiliases/metabolismo , Coenzimas/metabolismo , Mononucleotídeo de Flavina/metabolismo , Aspergillus niger/enzimologia , Sítios de Ligação , Carboxiliases/genética , Coenzimas/química , Estabilidade Enzimática , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mononucleotídeo de Flavina/química , Espectrometria de Massas/métodos , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5'-GUN-3' tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We report the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbors a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveals an unexpected diversity in the reductive chemistry catalyzed by these enzymes.
Assuntos
Nucleosídeo Q/análogos & derivados , Nucleosídeo Q/biossíntese , Oxirredutases/química , RNA de Transferência/química , Streptococcus thermophilus/enzimologia , Vitamina B 12/química , Sequência de Aminoácidos , Catálise , Cobalto/química , Cristalografia por Raios X , Halogenação , Dados de Sequência Molecular , Nucleosídeo Q/química , Oxirredução , Oxirredutases/genética , Estrutura Secundária de Proteína , SoluçõesRESUMO
Ubiquinone (also known as coenzyme Q) is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor. Despite structural and biochemical characterization of UbiX as a flavin mononucleotide (FMN)-binding protein, no decarboxylase activity has been detected. Here we report that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyltransferase mechanism of UbiX resembles that of the terpene synthases. The active site environment is dominated by π systems, which assist phosphate-C1' bond breakage following FMN reduction, leading to formation of the N5-C1' bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3'-C6 bond. Our findings establish the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoires.