Search

Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2-). Previous chemical rescue studies identified a putative FeIII-O2- intermediate that precedes substrate oxidation in Mus musculus cysteine dioxygenase (Mm CDO). Given that a similar reactive intermediate has been identified in the extradiol dioxygenase 2, 3-HCPD, it is conceivable that these enzymes share other mechanistic features with regard to substrate oxidation. To explore this possibility, enzymatic reactions with Mm CDO (as well as the bacterial 3-mercaptopropionic acid dioxygenase, Av MDO) were performed using a substrate analogue (2-mercaptoaniline, 2ma). This aromatic thiol closely approximates the catecholic substrate of homoprotocatechuate of 2, 3-HPCD while maintaining the 2-carbon thiol-amine separation preferred by Mm CDO. Remarkably, both enzymes exhibit 2ma-gated O2-consumption; however, none of the expected products for thiol dioxygenase or intra/extradiol dioxygenase reactions were observed. Instead, benzothiazoles are produced by the condensation of 2ma with aldehydes formed by an off-pathway oxidation of primary alcohols added to aqueous reactions to solubilize the substrate. The observed oxidation of 1o-alcohols in 2ma-reactions is consistent with the formation of a high-valent intermediate similar to what has been reported for cytochrome P450 and mononuclear iron model complexes.

3-mercaptopropionate dioxygenase from Azotobacter vinelandii ( Av MDO) is a non-heme mononuclear iron enzyme that catalyzes the O 2 -dependent oxidation of 3-mercaptopropionate ( 3mpa ) to produce 3-sulfinopropionic acid ( 3spa ). With one exception, the active site residues of MDO are identical to bacterial cysteine dioxygenase (CDO). Specifically, the CDO Arg-residue (R50) is replaced by Gln (Q67) in MDO. Despite this minor active site perturbation, substrate-specificity of Av MDO is more relaxed as compared to CDO. In order to investigate the relative timing of chemical and non-chemical events in Av MDO catalysis, the pH/D-dependence of steady-state kinetic parameters ( k cat and k cat / K M ) and viscosity effects are measured using two different substrates [ 3mpa and l -cysteine ( cys )]. The pL-dependent activity of Av MDO in these reactions can be rationalized assuming a diprotic enzyme model in which three ionic forms of the enzyme are present [cationic, E (z+1) ; neutral, E z ; and anionic, E (z−1) ]. The activities observed for each substrate appear to be dominated by electrostatic interactions within the enzymatic active site. Given the similarity between MDO and the more extensively characterized mammalian CDO, a tentative model for the role of the conserved ‘ catalytic triad ’ is proposed.

Here, we report the very first example of half-site reactivity and negative cooperativity involving an important F420 cofactor-dependent enzyme. F420H2:NADP(+) oxidoreductase (Fno) is an F420 cofactor-dependent enzyme that catalyzes the reversible reduction of NADP(+) through the transfer of a hydride from the reduced F420 cofactor. These catalytic processes are of major significance in numerous biochemical processes. While the steady-state kinetic analysis showed classic Michaelis-Menten kinetics with varying concentrations of the F420 redox moiety, FO, such plots revealed non-Michaelis-Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying concentrations of NADPH displays a downward concave shape, suggesting that negative cooperativity occurs between the two identical monomers. The transient state kinetic data show a burst prior to entering steady-state turnover. The burst suggests that product release is rate-limiting, and the amplitude of the burst phase corresponds to production of product in only one of the active sites of the functional dimer. These results suggest either half-site reactivity or an alternate sites model wherein the reduction of the cofactor, FO occurs at one active site at a time followed by reduction at the second active site. Thus, the data imply that Fno may be a functional regulatory enzyme.

F420 and FO are phenolic 5-deazaflavin cofactors that complement nicotinamide and flavin redox coenzymes in biochemical oxidoreductases and photocatalytic systems. Specifically, these 5-deazaflavins lack the single electron reactivity with O2 of riboflavin-derived coenzymes (FMN and FAD), and, in general, have a more negative redox potential than NAD(P)(+). For example, F420-dependent NADP(+) oxidoreductase (Fno) is critical to the conversion of CO2 to CH4 by methanogenic archaea, while FO functions as a light-harvesting agent in DNA repair. The preparation of these cofactors is an obstacle to their use in biochemical studies and biotechnology. Here, a convenient synthesis of FO was achieved by improving the redox stability of synthetic intermediates containing a polar, electron-rich aminophenol fragment. Improved yields and simplified purification techniques for FO are described. Additionally, Fno activity was restored with FO in the absence of F420. Investigating the FO-dependent NADP(+)/NADPH redox process by stopped-flow spectrophotometry, steady state kinetics were defined as having a Km of 4.00 ± 0.39 μM and a kcat of 5.27 ± 0.14 s(-1). The preparation of FO should enable future biochemical studies and novel uses of F420 mimics.

Post-transcriptional modifications of tRNA are made to structurally diversify tRNA. These modifications alter noncovalent interactions within the ribosomal machinery, resulting in phenotypic changes related to cell metabolism, growth, and virulence. MiaE is a carboxylate bridged, nonheme diiron monooxygenase, which catalyzes the O2-dependent hydroxylation of a hypermodified-tRNA nucleoside at position 37 (2-methylthio-N(6)-isopentenyl-adenosine(37)-tRNA) [designated ms(2)i(6)A37]. In this work, recombinant MiaE was cloned from Salmonella typhimurium , purified to homogeneity, and characterized by UV-visible and dual-mode X-band EPR spectroscopy for comparison to other nonheme diiron enzymes. Additionally, three nucleoside substrate-surrogates (i(6)A, Cl(2)i(6)A, and ms(2)i(6)A) and their corresponding hydroxylated products (io(6)A, Cl(2)io(6)A, and ms(2)io(6)A) were synthesized to investigate the chemo- and stereospecificity of this enzyme. In the absence of the native electron transport chain, the peroxide-shunt was utilized to monitor the rate of substrate hydroxylation. Remarkably, regardless of the substrate (i(6)A, Cl(2)i(6)A, and ms(2)i(6)A) used in peroxide-shunt assays, hydroxylation of the terminal isopentenyl-C4-position was observed with >97% E-stereoselectivity. No other nonspecific hydroxylation products were observed in enzymatic assays. Steady-state kinetic experiments also demonstrate that the initial rate of MiaE hydroxylation is highly influenced by the substituent at the C2-position of the nucleoside base (v0/[E] for ms(2)i(6)A > i(6)A > Cl(2)i(6)A). Indeed, the >3-fold rate enhancement exhibited by MiaE for the hydroxylation of the free ms(2)i(6)A nucleoside relative to i(6)A is consistent with previous whole cell assays reporting the ms(2)io(6)A and io(6)A product distribution within native tRNA-substrates. This observation suggests that the nucleoside C2-substituent is a key point of interaction regulating MiaE substrate specificity.

Electrospray ionization-mass spectrometry (ESI-MS) was used to investigate stereoselective interactions between seven zwitterionic alkylsulfonate-modified cinchona alkaloid chiral selectors and biologically relevant lysine-alanine-alanine tripeptide and alanine-alanine dipeptide selectands in modified methanolic solutions. Ion intensities from full scan mass spectra were used to assess degrees of association, the ratios of which were used to calculate selectivities for different selector-selectand pairs. The results support prior work on similar systems using HPLC, in that binding is mediated in these systems primarily through the quinuclidine amine on the selector and the C-terminal carboxylate of the peptide. N(α)- and N(α), N(ɛ)-acetylated forms of the tripeptide were used to study the relative contribution to binding imparted by the presence of multiple basic amines on the tripeptide with the selectors; this was not previously investigated by HPLC. The ability of the sulfonate group on the selector to reach and preferentially interact with the N(ɛ)-amine on the side chain of lysine was revealed. Overall, in acidic methanol conditions (0.5% acetic acid), degrees of association ranged from 1.5% to 17%, and selectivities ranged from non-selective to a 5.5:1 preference for binding one peptide stereoisomer over another with a given chiral selector. In sodium acetate (100 μM)-modified methanol solutions, significant changes in degrees of association (ranging from 4% to 25%) and selectivities (ranging from non-selective to 4.2:1 preference) were observed. These mass spectrometry experiments help to clarify the chiral recognition mechanism for these selectors and suggest that retention and selectivity could be further modulated in HPLC experiments through the utilization of alkali salt-containing mobile phases.

A mild, operationally simple, and single-step transition-metal-free protocol for the synthesis of enantiomerically pure (R)-(+)-2'-amino-1,1'-binaphthalen-2-ol (R-NOBIN) from (R)-(+)-1,1'-binaphthyl-2,2'-diamine (R-BINAM) is reported. The one-pot conversion proceeds with good yield and shows no racemization. The hydroxyl on the R-NOBIN product was shown to have come from water in the reaction medium via an H2(18)O study. The correct value of the specific rotation of R-NOBIN was reported.

In the search for bioactive sphingosine 1-phosphate (S1P) receptor ligands, a series of 2-amino-2-heterocyclic-propanols were synthesized. These molecules were discovered to be substrates of human-sphingosine kinases 1 and 2 (SPHK1 and SPHK2). When phosphorylated, the resultant phosphates showed varied activities at the five sphingosine-1-phosphate (S1P) receptors (S1P(1-5)). Agonism at S1P(1) was displayed in vivo by induction of lymphopenia. A stereochemical preference of the quaternary carbon was crucial for phosphorylation by the kinases and alters binding affinities at the S1P receptors. Oxazole and oxadiazole compounds are superior kinase substrates to FTY720, the prototypical prodrug immunomodulator, fingolimod (FTY720). The oxazole-derived structure was the most active for human SPHK2. Imidazole analogues were less active substrates for SPHKs, but more potent and selective agonists of the S1P(1) receptor; additionally, the imidazole class of compounds rendered mice lymphopenic.

Trovafloxacin (Trovan) is a fluoroquinolone antibiotic drug with a long half-life and broad-spectrum activity. Since its entry into the market in 1998, trovafloxacin has been associated with numerous cases of hepatotoxicity, which has limited its clinical usefulness. Trovafloxacin possesses two substructural elements that have the potential to generate reactive intermediates: a cyclopropylamine moiety and a difluoroanilino system. The results presented here describe the in vitro metabolic activation of a synthetic drug model (DM) of trovafloxacin that contains the cyclopropylamine moiety. Cyclopropylamine can be oxidized to reactive ring-opened products-a carbon-centered radical and a subsequently oxidized alpha,beta-unsaturated aldehyde. Experiments with monoamine oxygenases, horseradish peroxidase, flavin monooxygenase 3, and cDNA-expressed P450 isoenzymes revealed that P450 1A2 oxidizes DM to a reactive alpha,beta-unsaturated aldehyde, M 1. Furthermore, myeloperoxidase (MPO) was also demonstrated to oxidize DM in the presence of chloride ion to produce M 1. DM proved to be a suicide inhibitor of MPO while showing no inhibition of P450 1A2. The structure of the reactive metabolite was confirmed by LC-MS/MS analysis by comparison with a synthetic standard. M 1 was further shown to react with glutathione and the related thiol nucleophile, 4-bromobenzyl mercaptan, suggesting the potential of this intermediate to react with protein nucleophiles. In summary, these data provide evidence that trovafloxacin-induced hepatotoxicity may be mediated through the oxidation of the cyclopropylamine substructure to reactive intermediates that may form covalent adducts to hepatic proteins, resulting in damage to liver tissue.

The mechanism for the hepatotoxicity of trovafloxacin remains unresolved. Trovafloxacin contains a cyclopropylamine moiety which has a potential to be oxidized to reactive intermediate(s) although other putative elements may exist. In this study, a drug model of trovafloxacin containing the cyclopropylamine substructure was synthesized. Chemical oxidation of the drug model by K(3)Fe(CN)(6) and NaClO revealed that both oxidants oxidize this drug model to a reactive alpha,beta-unsaturated aldehyde, 11. The structure of 11 was fully elucidated by LC/MS/MS and NMR analysis. These results suggested that P450s with heme-iron center and myeloperoxidase generating hypochlorous acid in the presence of chloride ion are capable of bioactivating the cyclopropylamine moiety of trovafloxacin. This deleterious metabolism may lead to eventual hepatotoxicity.

MiaE [2-methylthio-N(6)-isopentenyl-adenosine(37)-tRNA monooxygenase] isolated from Salmonella typhimurium is a unique non-heme diiron enzyme that catalyzes the O2-dependent post-transcriptional allylic hydroxylation of a hypermodified nucleotide (ms(2)i(6)A37) at position 37 of selected tRNA molecules to produce 2-methylthio-N(6)-(4-hydroxyisopentenyl)-adenosine(37). In this work, isopentenylated tRNA substrates for MiaE were produced from small RNA oligomers corresponding to the anticodon stem loop (ACSL) region of tRNA(Trp) using recombinant MiaA and dimethylallyl pyrophosphate. Steady-state rates for MiaE-catalyzed substrate hydroxylation were determined using recombinant ferredoxin (Fd) and ferredoxin reductase (FdR) to provide a catalytic electron transport chain (ETC) using NADPH as the sole electron source. As with previously reported peroxide-shunt assays, steady-state product formation retains nearly stoichiometric (>98%) E stereoselectivity. MiaE-catalyzed i(6)A-ACSL(Trp) hydroxylation follows Michaelis-Menten saturation kinetics with kcat, KM, and V/K determined to be 0.10 ± 0.01 s(-1), 9.1 ± 1.5 μM, and ∼11000 M(-1) s(-1), respectively. While vastly slower, MiaE-catalyzed hydroxylation of free i(6)A nucleoside could also be observed using the (Fd/FdR)-ETC assay. By comparison to the V/K determined for i(6)A-ACSL substrates, an ∼6000-fold increase in enzymatic efficiency is imparted by ACSL(Trp)-MiaE interactions. The impact of substrate tRNA-MiaE interactions on protein secondary structure and active site electronic configuration was investigated using circular dichroism, dual-mode X-band electron paramagnetic resonance, and Mossbauer spectroscopies. These studies demonstrate that binding of tRNA to MiaE induces a protein conformational change that influences the electronic structure of the diiron site analogous to what has been observed for various bacterial multicomponent diiron monooxygenases upon titration with their corresponding effector proteins. These observations suggest that substrate-enzyme interactions may play a pivotal role in modulating the reactivity of the MiaE diiron active site. Moreover, the simplified monomeric (α) protein configuration exhibited by MiaE provide an unparalleled opportunity to study the impact of protein-effector interactions on non-heme diiron site geometry and reactivity.

Molecules such as benzene, that have six pi electrons cyclically delocalized, have the special stability that chemists call aromaticity. When electricity passes through a molecular wire containing a benzene ring a quinone-like structure is induced, and there is some loss of aromaticity. The conductance of such systems correlates with the magnitude of this effect. A molecule such as cyclobutadiene with only four cyclically delocalized pi electrons has special instability, called antiaromaticity. Evidence that such antiaromatic systems could convey high conductance in appropriate systems is derived from electrochemical studies, which also detect geometric changes in some thiophene-derived nanolength wires.