A two-component flavin-dependent monooxygenase involved in actinorhodin biosynthesis in Streptomyces coelicolor

The two-component flavin-dependent monooxygenases belong to an emerging class of enzymes involved in oxidation reactions in a number of metabolic and biosynthetic pathways in microorganisms. One component is a NAD(P)H:flavin oxidoreductase, which provides a reduced flavin to the second component, the proper monooxygenase. There, the reduced flavin activates molecular oxygen for substrate oxidation. Here, we study the flavin reductase ActVB and ActVA-ORF5 gene product, both reported to be involved in the last step of biosynthesis of the natural antibiotic actinorhodin in Streptomyces coelicolor. For the first time we show that ActVA-ORF5 is a FMN-dependent monooxygenase that together with the help of the flavin reductase ActVB catalyzes the oxidation reaction. The mechanism of the transfer of reduced FMN between ActVB and ActVA-ORF5 has been investigated. Dissociation constant values for oxidized and reduced flavin (FMNox and FMNred) with regard to ActVB and ActVA-ORF5 have been determined. The data clearly demonstrate a thermodynamic transfer of FMNred from ActVB to ActVA-ORF5 without involving a particular interaction between the two protein components. In full agreement with these data, we propose a reaction mechanism in which FMNox binds to ActVB, where it is reduced, and the resulting FMNred moves to ActVA-ORF5, where it reacts with O2 to generate a flavinperoxide intermediate. A direct spectroscopic evidence for the formation of such species within ActVA-ORF5 is reported.     

Oxidation of 2-thioflavins by peroxides. Formation of flavin 2-S-oxides

The reaction of 2-thioriboflavin (sulfur replacing the oxygen substituent at position 2 alpha) with hydrogen peroxide at pH approximately 10 leads to a blue flavin (lambda max = 565 nm) which was purified in stable, homogeneous form. Titrations of 2-thioflavins with m-chloroperoxybenzoic acid also yield the same blue flavins with consumption of 1 eq of peracid. Anaerobic reduction of the blue flavin by sodium dithionite requires 4e- eq, and leads to formation of 1,5-dihydro-2-thioflavin. Oxidation of the latter with O2 restores the original 2-thioflavin. pH titration of the blue flavin shows two pKa values of 2.4 and 6.6, with no apparent ionization in the pH range 8-11. These results suggest that the blue flavin is a flavin 2-S-oxide. The visible absorption spectra of flavin 2-S-oxides show a pronounced dependence on solvent polarity. This property suggests that these flavin analogs may be useful hydrophilic/hydrophobic probes of flavoprotein active sites. Flavin 2-S-oxides can be oxidized further to the 2-sulfinate and 2-sulfonate analogs, some properties of which are described.     

Mechanism of flavin reduction in the alkanesulfonate monooxygenase system

The alkanesulfonate monooxygenase system from Escherichia coli is involved in scavenging sulfur from alkanesulfonates under sulfur starvation. An FMN reductase (SsuE) catalyzes the reduction of FMN by NADPH, and the reduced flavin is transferred to the monooxygenase (SsuD). Rapid reaction kinetic analyses were performed to define the microscopic steps involved in SsuE catalyzed flavin reduction. Results from single-wavelength analyses at 450 and 550 nm showed that reduction of FMN occurs in three distinct phases. Following a possible rapid equilibrium binding of FMN and NADPH to SsuE (MC-1) that occurs before the first detectable step, an initial fast phase (241 s(-1)) corresponds to the interaction of NADPH with FMN (CT-1). The second phase is a slow conversion (11 s(-1)) to form a charge-transfer complex of reduced FMNH(2) with NADP(+) (CT-2), and represents electron transfer from the pyridine nucleotide to the flavin. The third step (19 s(-1)) is the decay of the charge-transfer complex to SsuE with bound products (MC-2) or product release from the CT-2 complex. Results from isotope studies with [(4R)-(2)H]NADPH demonstrates a rate-limiting step in electron transfer from NADPH to FMN, and may imply a partial rate-limiting step from CT-2 to MC-2 or the direct release of products from CT-2. While the utilization of flavin as a substrate by the alkanesulfonate monooxygenase system is novel, the mechanism for flavin reduction follows an analogous reaction path as standard flavoproteins.     

Stereochemistry and accessibility of prosthetic groups in flavoproteins

Using 8-demethyl-8-hydroxy-5-deaza-5-carba analogues of the appropriate flavin nucleotides, we determined the stereochemistry of interaction between coenzyme and substrate for several flavoproteins. The enzymes were D-amino acid oxidase, L-lactate oxidase, and D-lactate dehydrogenase, all three of which interact with pyruvate, as well as cyclohexanone monooxygenase and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase, which were both probed with nicotinamide nucleotides. L-Lactate oxidase and D-lactate dehydrogenase used the si face of the modified flavin ring while the other three enzymes showed re-side specificity. This selection of flavoenzymes includes FAD- and FMN-dependent enzymes, enzymes that follow a carbanion mechanism, and others that have hydride transfer as an integral part of their reaction pathway.     

Mechanism of flavin transfer and oxygen activation by the two-component flavoenzyme styrene monooxygenase

Styrene monooxygenase (SMO) from Pseudomonas putida S12 is a two-component flavoenzyme composed of the NADH-specific flavin reductase, SMOB, and FAD-specific styrene epoxidase, SMOA. Here, we report the cloning, and expression of native and histidine-tagged versions of SMOA and SMOB and studies of the flavin transfer and styrene oxygenation reactions. In the reductive half-reaction, SMOB catalyzes the two-electron reduction of FAD with a turnover number of 3200 s(-1). Single turnover studies of the reaction of reduced SMOA with substrates indicate the formation of a stable oxygen intermediate with the absorbance characteristics of a flavin hydroperoxide. Based on the results of numerical simulations of the steady-state mechanism of SMO, we find that the observed coupling of NADH and styrene oxidation can be best explained by a model, which includes both the direct transfer and passive diffusion of reduced FAD from SMOB to SMOA.     

Altered mechanism of the alkanesulfonate FMN reductase with the monooxygenase enzyme

The two-component alkanesulfonate monooxygenase system from Escherichia coli is comprised of an FMN reductase (SsuE) and a monooxygenase enzyme (SsuD) that together catalyze the oxidation of alkanesulfonate to the corresponding aldehyde and sulfite products. To determine the effects of protein interactions on catalysis, the steady-state kinetic parameters for SsuE were determined in single-enzyme assays and in the presence of the monooxygenase enzyme and alkanesulfonate substrate. In single-enzyme kinetic assays, SsuE followed an ordered sequential mechanism, with NADPH as the first substrate to bind and NADP+ as the last product to dissociate. However, in the presence of SsuD and octanesulfonate the kinetic mechanism of SsuE is altered to a rapid equilibrium ordered mechanism, and the Km value for FMN is increased 10-fold. These results suggest that both the SsuD enzyme and alkanesulfonate substrate are required to ensure that the FMN reductase reaction proceeds to form the ternary complex with the subsequent generation of reduced flavin transfer.     

Identity of the emitter in the bacterial luciferase luminescence reaction: binding and fluorescence quantum yield studies of 5-decyl-4a-hydroxy-4a,5-dihydroriboflavin-5'-phosphate as a model

The excited state of 4a-hydroxy-4a,5-dihydroFMN has been postulated to be the emitter in the bacterial bioluminescence reaction. However, while the bioluminescence quantum yield of the luciferase emitter is about 0.16, chemiluminescence and fluorescence quantum yields of earlier flavin models mimicking the luciferase emitter were no more than 10(-5). To further examine the proposed chemical identity of the luciferase emitter, 5-decyl-4a-hydroxy-4a,5-dihydroFMN was prepared as a new flavin model. Both the wild-type Vibrio harveyi luciferase and a catalytically active alphaC106A mutant formed complexes with the flavin model at a 1:1 molar ratio with K(d) values at 2.4 and 1.2 microM, respectively. This flavin model inhibited the activity of both luciferases, suggesting that it was bound to the enzyme active center. While the free flavin model was itself only very weakly fluorescent, its binding to either luciferase species resulted in markedly enhanced fluorescence, peaking at 440 nm. The fluorescence quantum yields of 5-decyl-4a-hydroxy-4a,5-dihydroFMN bound to wild-type and alphaC106A luciferases were 0.08 and 0.05, respectively, which are about 50% of the respective emitter bioluminescence quantum yields of these two luciferases. The present findings clearly demonstrated that the luciferase active site was suitable for marked enhancement of fluorescence of 4a-hydroxyflavin and, hence, provides a strong support to the proposed identity of 4a-hydroxy-4a,5-dihydroFMN, in its exited state, as the luciferase emitter.     

Mechanistic studies of cyclohexanone monooxygenase: chemical properties of intermediates involved in catalysis

Cyclohexanone monooxygenase (CHMO), a bacterial flavoenzyme, carries out an oxygen insertion reaction on cyclohexanone to form a seven-membered cyclic product, epsilon-caprolactone. The reaction catalyzed involves the four-electron reduction of O2 at the expense of a two-electron oxidation of NADPH and a two-electron oxidation of cyclohexanone to form epsilon-caprolactone. Previous studies suggested the participation of either a flavin C4a-hydroperoxide or a flavin C4a-peroxide intermediate during the enzymatic catalysis [Ryerson, C. C., Ballou, D. P., and Walsh, C. (1982) Biochemistry 21, 2644-2655]. However, there was no kinetic or spectral evidence to distinguish between these two possibilities. In the present work we used double-mixing stopped-flow techniques to show that the C4a-flavin-oxygen adduct, which is formed rapidly from the reaction of oxygen with reduced enzyme in the presence of NADP, can exist in two states. When the reaction is carried out at pH 7.2, the first intermediate is a flavin C4a-peroxide with maximum absorbance at 366 nm; this intermediate becomes protonated at about 3 s(-1) to form what is believed to be the flavin C4a-hydroperoxide with maximum absorbance at 383 nm. These two intermediates can be interconverted by altering the pH, with a pK(a) of 8.4. Thus, at pH 9.0 the flavin C4a-peroxide persists mainly in the deprotonated form. Further kinetic studies also demonstrated that only the flavin C4a-peroxide intermediate could oxygenate the substrate, cyclohexanone. The requirement in catalysis of the deprotonated flavin C4a-peroxide, a nucleophile, is consistent with a Baeyer-Villiger rearrangement mechanism for the enzymatic oxygenation of cyclohexanone. In the course of these studies, the Kd for cyclohexanone to the C4a-peroxyflavin form of CHMO was determined to be approximately 1 microM. The rate-determining step in catalysis was shown to be the release of NADP from the oxidized enzyme.     

Luminol chemiluminescence: Chemistry,excitation, emitter

The mechanism of luminol chemiluminescence is a special case of nucleophilic addition to carbonyl compounds. The breakdown of the key intermediate, an alpha hydroxy hydroperoxide, produces a peracid ortho to an acyl diazene group. After intramolecular addition of the peracid, the energy from nitrogen expulsion is utilized in the formation of an anti-aromatic endoperoxide. Rupture along the O,O bond leaves a substantial part of the ensuing phthalate in its excited state. The emitter is shown to be a mono-protonated phthalate unaccessible by photoexcitation. The dark reaction is a concerted decomposion of the alpha hydroxy hydroperodixe to yield ground-state phthalate.     

Mechanism for sulfur acquisition by the alkanesulfonate monooxygenase system

The bacterial alkanesulfonate monooxygenase system is involved in the acquisition of sulfur from organosulfonated compounds during limiting sulfur conditions. The reaction relies on an FMN reductase to supply reduced flavin to the monooxygenase enzyme. The reaction catalyzed by the alkanesulfonate monooxygenase enzyme involves the carbon-sulfur bond cleavage of a wide range of organosulfonated compounds. A C4a-(hydro)peroxyflavin is the oxygenating intermediate in the mechanism of desulfonation by the alkanesulfonate monooxygenase. This review discusses the physiological importance of this system, and the individual kinetic parameters and mechanistic properties of this enzyme system.     

The enigmatic reaction of flavins with oxygen

The reaction of flavoenzymes with oxygen remains a fascinating area of research because of its relevance for reactive oxygen species (ROS) generation. Several exciting recent studies provide consistent mechanistic clues about the specific functional and structural properties of the oxidase and monooxygenase flavoenzymatic systems. Specifically, the spatial arrangement of the reacting oxygen that is in direct contact with the flavin group is emerging as a crucial factor that differentiates between oxidase and monooxygenase enzymes. A challenge for the future will be to use these emerging concepts to rationally engineer flavoenzymes, paving the way to new research avenues with far-reaching implications for oxidative biocatalysis and metabolic engineering.     

Structural and chemical trapping of flavin‐oxide intermediates reveals substrate‐directed reaction multiplicity

Though reactive flavin‐N5/C4α‐oxide intermediates can be spectroscopically profiled for some flavin‐assisted enzymatic reactions, their exact chemical configurations are hardly visualized. Structural systems biology and stable isotopic labelling techniques were exploited to correct this stereotypical view. Three transition‐like complexes, the α‐ketoacid…N5‐FMN_ox complex ( I ), the FMN_ox‐N5‐aloxyl‐C′α^?‐C4α⁺ zwitterion ( II ), and the FMN‐N5‐ethenol‐N5‐C4α‐epoxide ( III ), were determined from mandelate oxidase (Hmo) or its mutant Y128F (monooxygenase) crystals soaked with monofluoropyruvate (a product mimic), establishing that N5 of FMN_ox an alternative reaction center can polarize to an ylide‐like mesomer in the active site. In contrast, four distinct flavin‐C4α‐oxide adducts ( IV – VII ) from Y128F crystals soaked with selected substrates materialize C4α of FMN an intrinsic reaction center, witnessing oxidation, Baeyer–Villiger/peroxide‐assisted decarboxylation, and epoxidation reactions. In conjunction with stopped‐flow kinetics, the multifaceted flavin‐dependent reaction continuum is physically dissected at molecular level for the first time.     

Structures of the alkanesulfonate monooxygenase MsuD provide insight into C–S bond cleavage,substrate scope,and an unexpected role for the tetramer

Bacterial two-component flavin-dependent monooxygenases cleave the stable C–S bond of environmental and anthropogenic organosulfur compounds. The monooxygenase MsuD converts methanesulfonate (MS⁻) to sulfite, completing the sulfur assimilation process during sulfate starvation, but the mechanism of this conversion remains unclear. To explore the mechanism of C–S bond cleavage, we report a series of crystal structures of MsuD from Pseudomonas fluorescens in different liganded states. This report provides the first crystal structures of an alkanesulfonate monooxygenase with a bound flavin and alkanesulfonate, elucidating the roles of the active site lid, the protein C terminus, and an active site loop in flavin and/or alkanesulfonate binding. These structures position MS⁻ closest to the flavin N5 position, consistent with an N5-(hydro)peroxyflavin mechanism rather than a classical C4a-(hydro)peroxyflavin mechanism. A fully enclosed active site is observed in the ternary complex, mediated by interchain interaction of the C terminus at the tetramer interface. These structures identify an unexpected function of the protein C terminus in this protein family in stabilizing tetramer formation and the alkanesulfonate-binding site. Spurred by interest from the crystal structures, we conducted biochemical assays and molecular docking that redefine MsuD as a small- to medium-chain alkanesulfonate monooxygenase. Functional mutations verify the sulfonate-binding site and reveal the critical importance of the protein C terminus for monooxygenase function. These findings reveal a deeper understanding of MsuD’s functionality at the molecular level and consequently how it operates within its role as part of the sulfur assimilation pathway.     

Bacterial bioluminescence in vivo: control and synthesis of aldehyde factor in temperature-conditional luminescence mutants

Bioluminescent marine bacteria possess luciferase, which catalyzes the oxidation of reduced flavin mononucleotide and long-chain aldehyde to produce light. Temperature-sensitive mutants of these bacteria can be obtained which require exogenous aldehyde for light production at higher temperatures. In Beneckea harveyi. two classes of such mutants were found which differed with regard to their response to temperature shifts. In one class, a shift from permissive to nonpermissive temperature in liquid cultures resulted in a rapid (t((1/2)) approximately 3 min) loss of luminescence. In the other, there was no immediate decline in luminescence; it was the increase of luminescence that was blocked. Through studies of these and other effects of temperature shifts on the in vivo luminescence of these mutants, we conclude that at least two genes are specifically involved in the in vivo biosynthesis of aldehyde for the luminescence reaction and that both genes are coordinately controlled with that for luciferase.     

Catalytic importance of the substrate binding order for the FMNH2-dependent alkanesulfonate monooxygenase enzyme

The two-component alkanesulfonate monooxygenase system from Escherichia coli includes an FMN reductase (SsuE) and an FMNH2-dependent alkanesulfonate monooxygenase (SsuD) involved in the acquisition of sulfur from alkanesulfonates during sulfur starvation. The SsuD enzyme directly catalyzes the oxidation of alkanesulfonate to aldehyde and sulfite in the presence of O2 and FMNH2. The goal of these studies was to investigate the kinetic mechanism of SsuD through rapid reaction kinetics and substrate binding studies. The SsuD enzyme shows a clear preference for FMNH2 (Kd, 0.32 +/- 0.15 microM) compared to FMN (Kd, 10.2 +/- 0.4 microM) with a 1:1 binding stoichiometry for each form of the flavin. The kinetic trace of premixed SsuD and FMNH2 mixed with oxygenated buffer was best fit to a double exponential with no observed formation of the C4a-(hydro)peroxyflavin. However, when FMNH2 was mixed with SsuD and oxygenated buffer an initial fast phase (kobs, 12.9 s-1) was observed, suggesting that the mixing order is critical for the accumulation of the C4a-(hydro)peroxyflavin. Results from fluorimetric titrations with octanesulfonate imply that reduced flavin must bind first to promote octanesulfonate binding. When octanesulfonate was included in the kinetic studies the C4a-(hydro)peroxyflavin was observed at 370 nm when FMNH2 was not premixed with SsuD, which correlated with an increase in octanal product. There was a clear hyperbolic dependence on octanesulfonate binding, indicating that octanesulfonate binds in rapid equilibrium, and further results indicated there was a second isomerization step following binding. These results suggest that an ordered substrate binding mechanism is important in the desulfonation reaction by SsuD with reduced flavin binding first followed by either O2 or octanesulfonate.     

LuxG is a functioning flavin reductase for bacterial luminescence

The luxG gene is part of the lux operon of marine luminous bacteria. luxG has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study, luxG from Photobacterium leiognathi TH1 was cloned and expressed in Escherichia coli in both native and C-terminal His₆-tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of LuxG were purified to homogeneity, and their biochemical properties were characterized. Purified LuxG is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the LuxG reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4°C pH 8.0, K_m for NADH, K_m for FMN, and k_cat were calculated to be 15.1 μM, 2.7 μM, and 1.7 s⁻¹, respectively. Coupled assays between LuxG and luciferases from P. leiognathi TH1 and Vibrio campbellii also showed that LuxG could supply FMNH⁻ for light emission in vitro. A luxG gene knockout mutant of P. leiognathi TH1 exhibited a much dimmer luminescent phenotype compared to the native P. leiognathi TH1, implying that LuxG is the most significant source of FMNH⁻ for the luminescence reaction in vivo.     

Deletional studies to investigate the functional role of a dynamic loop region of alkanesulfonate monooxygenase

Several bacterial organisms rely on the two-component alkanesulfonate monooxygenase system for the acquisition of organosulfonate compounds when inorganic sulfur is limiting in the environment. This system is comprised of an FMN reductase (SsuE) that supplies reduced flavin to the alkanesulfonate monooxygenase (SsuD). Desulfonation of alkanesulfonates by SsuD is catalyzed through the activation of dioxygen by reduced flavin. The three-dimensional structure of SsuD exists as a TIM-barrel fold with several discrete insertion regions. An extensive insertion region near the putative active site was disordered in the SsuD structure, suggesting the importance of protein dynamics in the desulfonation mechanism. Three variants containing a partial deletion of the loop region were constructed to evaluate the functional properties of this region. There were no overall gross changes in secondary structure for the three SsuD deletion variants compared to wild-type SsuD, but each variant was found to be catalytically inactive. The deletion variants were unable to undergo the conformational changes necessary for catalysis even though they were able to bind reduced flavin. Rapid kinetic analyses monitoring the reductive and oxidative half-reactions indicated that the SsuD deletion variants failed to protect reduced flavin from unproductive oxidation. These studies define the importance of dynamic loop region for protection and stabilization of reduced flavin and reaction intermediates.     

Cloning and characterization of a gene cluster for cyclododecanone oxidation in Rhodococcus ruber SC1

Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cell. Rhodococcus ruber strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone as the sole carbon source. A reverse genetic approach was used to isolate a 10-kb gene cluster containing all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxidation were only marginally similar to the analogous genes for cyclohexanone oxidation. The biochemical function of the enzymes encoded on the 10-kb gene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol dehydrogenase, and the aldehyde dehydrogenase, was determined in Escherichia coli based on the ability to convert cyclododecanone. Recombinant E. coli strains grown in the presence of cyclododecanone accumulated lauryl lactone, 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyclododecanone monooxygenase is a type 1 Baeyer-Villiger flavin monooxygenase (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C₁₁ to C₁₅), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C₅ to C₇).     

Detection of protein-protein interactions in the alkanesulfonate monooxygenase system from Escherichia coli

The two-component alkanesulfonate monooxygenase system utilizes reduced flavin as a substrate to catalyze a unique desulfonation reaction during times of sulfur starvation. The importance of protein-protein interactions in the mechanism of flavin transfer was analyzed in these studies. The results from affinity chromatography and cross-linking experiments support the formation of a stable complex between the flavin mononucleotide (FMN) reductase (SsuE) and monooxygenase (SsuD). Interactions between the two proteins do not lead to overall conformational changes in protein structure, as indicated by the results from circular dichroism spectroscopy in the far-UV region. However, subtle changes in the flavin environment of FMN-bound SsuE that occur in the presence of SsuD were identified by circular dichroism spectroscopy in the visible region. These data are supported by the results from fluorescent spectroscopy experiments, where a dissociation constant of 0.0022 +/- 0.0010 muM was obtained for the binding of SsuE to SsuD. Based on these studies, the stoichiometry for protein-protein interactions is proposed to involve a 1:1 monomeric association of SsuE with SsuD.     

ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase

Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.