ABSTRACT
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.
Subject(s)
Hydrolases , NADP , Rhodobacteraceae , Escherichia coli/genetics , NADP/metabolism , Oxygen/chemistry , Vitamin B 12/metabolism , Phenols/chemistry , Phenols/metabolism , Electron Spin Resonance Spectroscopy , Hydrolases/chemistry , Hydrolases/genetics , Hydrolases/isolation & purification , Hydrolases/metabolism , Rhodobacteraceae/enzymology , Rhodobacteraceae/genetics , Protein Structure, Tertiary , Models, Molecular , Maltose-Binding Proteins/genetics , Maltose-Binding Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Coenzymes/metabolismABSTRACT
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.
Subject(s)
Carboxy-Lyases , Models, Molecular , Carboxy-Lyases/chemistry , Carboxy-Lyases/metabolism , Flavin Mononucleotide/metabolism , Protein Structure, Tertiary , Structure-Activity Relationship , Substrate SpecificityABSTRACT
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.
Subject(s)
Carboxy-Lyases/chemistry , Carboxy-Lyases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Biocatalysis , Catalytic Domain , Cycloaddition Reaction , Decarboxylation , Flavin Mononucleotide/metabolism , Kinetics , Models, Molecular , Oxygen/pharmacology , Protein Domains , Protein Subunits/chemistry , Protein Subunits/metabolism , Solvents , Structure-Activity Relationship , Substrate Specificity , ViscosityABSTRACT
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.
ABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/metabolism , Aspergillus niger/enzymology , Carboxy-Lyases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Fungal Proteins/metabolism , Metabolic Engineering/methods , Biocatalysis , Escherichia coli/genetics , Fatty Acids, Unsaturated/metabolism , Polyenes/metabolism , Polyketide Synthases/metabolism , Pyrroles/metabolismABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , Escherichia coli/genetics , Gene Expression , Iron-Sulfur Proteins/genetics , Oxidoreductases/genetics , Vitamin B 12/chemistry , Bacillus megaterium/enzymology , Bacillus megaterium/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites , Biodegradation, Environmental , Cloning, Molecular , Enterobacteriaceae/enzymology , Enterobacteriaceae/genetics , Escherichia coli/enzymology , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Halogens/chemistry , Halogens/metabolism , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Kinetics , Models, Molecular , Nucleoside Q/analogs & derivatives , Nucleoside Q/chemistry , Nucleoside Q/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Phyllobacteriaceae/enzymology , Phyllobacteriaceae/genetics , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Streptococcus thermophilus/enzymology , Streptococcus thermophilus/genetics , Vibrio/enzymology , Vibrio/genetics , Vitamin B 12/metabolismABSTRACT
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.
ABSTRACT
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.
Subject(s)
Alkynes/metabolism , Carboxy-Lyases/metabolism , Propionates/metabolism , Alkynes/chemistry , Biocatalysis , Carboxy-Lyases/chemistry , Carboxy-Lyases/isolation & purification , Cycloaddition Reaction , Density Functional Theory , Models, Molecular , Molecular Conformation , Propionates/chemistryABSTRACT
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.
Subject(s)
Carboxy-Lyases/chemistry , Electron Spin Resonance Spectroscopy/methods , Escherichia coli Proteins/chemistry , Escherichia coli/metabolism , Carboxy-Lyases/isolation & purification , Carboxy-Lyases/metabolism , Decarboxylation , Escherichia coli Proteins/isolation & purification , Escherichia coli Proteins/metabolism , Flavin Mononucleotide/chemistry , Oxidation-Reduction , Prenylation , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
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.
Subject(s)
Aspergillus niger/enzymology , Carboxy-Lyases/metabolism , Dimethylallyltranstransferase/metabolism , Dinitrocresols/metabolism , Fungal Proteins/metabolism , Binding Sites , Decarboxylation , Diphosphates/metabolism , Prenylation , Terpenes/metabolismABSTRACT
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.
ABSTRACT
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.
ABSTRACT
A hybrid algal photosynthesis and ion exchange (HAPIX) process was developed that uses natural zeolite (chabazite) and wild type algae to treat high ammonium (NH4+) strength wastewater. In the HAPIX process, NH4+ is temporarily adsorbed from the liquid, which reduces the free ammonia (FA) concentration below the inhibitory level for algal growth. The slow release of adsorbed NH4+ subsequently supports the continuous growth of algae. In this study, a HAPIX reactor reduced NH4+-N concentrations in centrate from an anaerobic digester from 1180â¯mgâ¯L-1 to below 10â¯mgâ¯L-1 without dilution. Chabazite doses of 60â¯gâ¯L-1 produced more algal biomass, with higher protein and starch contents, than doses of 150â¯gâ¯L-1 and 250â¯gâ¯L-1. Approximately 67-70% of fatty acids in the algal biomass harvested from HAPIX reactors were unsaturated. A mathematical framework that couples a homogeneous surface diffusion model with a co-limitation algal kinetic growth model reasonably predicted the algal biomass production and NH4+-N concentrations in the HAPIX reactors. The HAPIX process has the potential to serve a two-fold purpose of high NH4+-N strength wastewater treatment and agricultural or commercial biopolymer production.
Subject(s)
Ammonium Compounds/metabolism , Chlorella/drug effects , Water Pollutants, Chemical/metabolism , Zeolites/pharmacology , Biomass , Bioreactors , Chlorella/growth & development , Chlorella/metabolism , Ion Exchange , Photosynthesis , Waste Disposal, Fluid , WastewaterABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , NADH, NADPH Oxidoreductases/metabolism , NADP/metabolism , Phyllobacteriaceae/enzymology , Bacterial Proteins/chemistry , Halogenation , Hydrogen-Ion Concentration , Models, Molecular , NADH, NADPH Oxidoreductases/chemistry , Osmolar Concentration , Oxidation-Reduction , Phyllobacteriaceae/chemistry , Phyllobacteriaceae/metabolismABSTRACT
Nitrification of high total ammonia nitrogen-strength wastewaters is challenging due to free ammonia (FA) inhibition of nitrification. FA inhibition can potentially be alleviated by temporarily adsorbing ammonium (NH4+) to natural zeolite, such as chabazite, followed by direct zeolite bioregeneration via nitrification. In this research, the effectiveness of chabazite addition for reducing nitrification inhibition during treatment of centrate from anaerobic digestion of swine waste was quantified. A mathematical model was developed that accounts for ion exchange of NH4+ and sodium at the chabazite surface, surface diffusion of adsorbed NH4+ within the chabazite grains, sequential nitrification of aqueous NH4+ to nitrite and nitrate, and inhibition of nitritation and nitratation rates by NH4+. The model was calibrated using results of abiotic ion exchange and nitrification studies. Subsequently, nitrification tests were carried out with synthetic wastewater with a NH4+-N concentration of 1000 mg L-1, with and without chabazite. A chabazite dose of 150 g L-1 decreased the FA concentration to below the inhibitory level and increased the nitrification rate from 0.16 to 0.36 mg-N (g-VSS)-1 h-1. Following calibration, the model could predict the experimental data with no additional fitting parameters or parameter adjustment, in both the presence and absence of chabazite. The results suggest that the mathematical model provides a theoretically sound conceptual understanding of ion exchange assisted nitrification.
Subject(s)
Nitrification , Zeolites , Animals , Bioreactors , Livestock , Nitrogen , Swine , Waste Disposal, Fluid , WastewaterABSTRACT
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.
Subject(s)
Bacillus megaterium/genetics , Chloroform/chemistry , Oxidoreductases/genetics , Halogenation , Recombinant Proteins/geneticsABSTRACT
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.
Subject(s)
Aspergillus niger/enzymology , Carboxy-Lyases/chemistry , Carboxy-Lyases/metabolism , Flavin Mononucleotide/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Aspergillus niger/chemistry , Aspergillus niger/genetics , Binding Sites , Carboxy-Lyases/genetics , Catalysis , Catalytic Domain , Conserved Sequence , Flavin Mononucleotide/chemistry , Fungal Proteins/genetics , Isomerism , Kinetics , Oxidation-ReductionABSTRACT
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.
Subject(s)
Carboxy-Lyases , Dimethylallyltranstransferase , Escherichia coli Proteins , Escherichia coli , Flavins , Flavoproteins , Prenylation/physiology , Carboxy-Lyases/chemistry , Carboxy-Lyases/genetics , Carboxy-Lyases/metabolism , Dimethylallyltranstransferase/chemistry , Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/metabolism , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Flavins/biosynthesis , Flavins/chemistry , Flavins/genetics , Flavoproteins/chemistry , Flavoproteins/genetics , Flavoproteins/metabolismABSTRACT
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.
Subject(s)
Carboxy-Lyases/metabolism , Escherichia coli/metabolism , Flavin Mononucleotide/metabolism , Ubiquinone/metabolism , Binding Sites , Carboxy-Lyases/chemistry , Crystallography, X-Ray , Electron Spin Resonance Spectroscopy , Electron Transport , Escherichia coli/chemistry , Models, Molecular , Oxidation-Reduction , Prenylation , Protein Domains , Ubiquinone/chemistryABSTRACT
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.