Donald F. Stec

The IsdG-family of haem oxygenases degrades haem to a novel chromophore

Enzymatic haem catabolism by haem oxygenases is conserved from bacteria to humans and proceeds through a common mechanism leading to the formation of iron, carbon monoxide and biliverdin. The first members of a novel class of haem oxygenases were recently identified in Staphylococcus aureus (IsdG and IsdI) and were termed the IsdG‐family of haem oxygenases. Enzymes of the IsdG‐family form tertiary structures distinct from those of the canonical haem oxygenase family, suggesting that IsdG‐family members degrade haem via a unique reaction mechanism. Herein we report that the IsdG‐family of haem oxygenases degrade haem to the oxo‐bilirubin chromophore staphylobilin. We also present the crystal structure of haem‐bound IsdI in which haem ruffling and constrained binding of oxygen is consistent with cleavage of the porphyrin ring at the β‐ or δ‐meso carbons. Combined, these data establish that the IsdG‐family of haem oxygenases degrades haem to a novel chromophore distinct from biliverdin.

Enzymatic synthesis of a bicyclobutane fatty acid by a hemoprotein lipoxygenase fusion protein from the cyanobacterium Anabaena PCC 7120.

Biological transformations of polyunsaturated fatty acids often lead to chemically unstable products, such as the prostaglandin endoperoxides and leukotriene A4 epoxide of mammalian biology and the allene epoxides of plants. Here, we report on the enzymatic production of a fatty acid containing a highly strained bicyclic four-carbon ring, a moiety known previously only as a model compound for mechanistic studies in chemistry. Starting from linolenic acid (C18.3ω3), a dual function protein from the cyanobacterium Anabaena PCC 7120 forms 9R-hydroperoxy-C18.3ω3 in a lipoxygenase domain, then a catalase-related domain converts the 9R-hydroperoxide to two unstable allylic epoxides. We isolated and identified the major product as 9R,10R-epoxy-11trans-C18.1 containing a bicyclo[1.1.0]butyl ring on carbons 13–16, and the minor product as 9R,10R-epoxy-11trans,13trans,15cis-C18.ω3, an epoxide of the leukotriene A type. Synthesis of both epoxides can be understood by initial transformation of the hydroperoxide to an epoxy allylic carbocation. Rearrangement to an intermediate bicyclobutonium ion followed by deprotonation gives the bicyclobutane fatty acid. This enzymatic reaction has no parallel in aqueous or organic solvent, where ring-opened cyclopropanes, cyclobutanes, and homoallyl products are formed. Given the capability shown here for enzymatic formation of the highly strained and unstable bicyclobutane, our findings suggest that other transformations involving carbocation rearrangement, in both chemistry and biology, should be examined for the production of the high energy bicyclobutanes.

Cooperativity in oxidation reactions catalyzed by cytochrome P450 1A2: highly cooperative pyrene hydroxylation and multiphasic kinetics of ligand binding.

Rabbit liver cytochrome P450 (P450) 1A2 was found to catalyze the 5,6-epoxidation of α-naphthoflavone (αNF), 1-hydroxylation of pyrene, and the subsequent 6-, 8-, and other hydroxylations of 1-hydroxy (OH) pyrene. Plots of steady-state rates of product formation versus substrate concentration were hyperbolic for αNF epoxidation but highly cooperative (Hill n coefficients of 2-4) for pyrene and 1-OH pyrene hydroxylation. When any of the three substrates (αNF, pyrene, 1-OH pyrene) were mixed with ferric P450 1A2 using stopped-flow methods, the changes in the heme Soret spectra were relatively slow and multiphasic. Changes in the fluorescence of all of the substrates were much faster, consistent with rapid initial binding to P450 1A2 in a manner that does not change the heme spectrum. For binding of pyrene to ferrous P450 1A2, the course of the spectra revealed sequential changes in opposite directions, consistent with P450 1A2 being involved in a series of transitions to explain the kinetic multiphasicity as opposed to multiple, slowly interconverting populations of enzyme undergoing the same event at different rates. Models of rabbit P450 1A2 based on a published crystal structure of a human P450 1A2-αNF complex show active site space for only one αNF or for two pyrenes. The spectral changes observed for binding and hydroxylation of pyrene and 1-OH pyrene could be fit to a kinetic model in which hydroxylation occurs only when two substrates are bound. Elements of this mechanism may be relevant to other cases of P450 cooperativity.

Synthesis of dihydroperoxides of linoleic and linolenic acids and studies on their transformation to 4-hydroperoxynonenal.

The cytotoxic aldehydes 4-hydroxynonenal, 4-hydroperoxynonenal (4-HPNE), and 4-oxononenal are formed during lipid peroxidation via oxidative transformation of the hydroxy or hydroperoxy precursor fatty acids, respectively. The mechanism of the carbon chain cleavage reaction leading to the aldehyde fragments is not known, but Hock-cleavage of a suitable dihydroperoxide derivative was implicated to account for the fragmentation [Schneider, C., Tallman, K.A., Porter, N.A., and Brash, A.R. (2001) Two Distinct Pathways of Formation of 4-Hydroxynonenal. Mechanisms of Nonenzymatic Transformation of the 9- and 13-Hydroperoxides of Linoleic Acid to 4-Hydroxyalkenals, J. Biol. Chem. 275, 20831–20838]. Both 8,13- and 10,13-dihydroperoxyoctadecadienoic acids (diHPODE) could serve as precursors in a Hock-cleavage leading to 4-HPNE via two different pathways. Here, we synthesized diastereomeric 9,12-, 10,12-, and 10,13-diHPODE using singlet oxidation of linoleic acid. 8,13-Dihydroperoxyoctadecatrienoic acid was synthesized by vitamin E-controlled autoxidation of γ-linolenic acid followed by reaction with soybean lipoxygenase. The transformation of these potential precursors to 4-HPNE was studied under conditions of autoxidation, hematin-, and acid-catalysis. In contrast to 9- or 13-HPODE, neither of the dihydroperoxides formed 4-HPNE on autoxidation (lipid film, 37°C), regardless of whether the free acid or the methyl ester derivative was used. Acid treatment of 10,13-diHPODE led to the expected formation of 4-HPNE as a significant product, in accord with a Hock-type cleavage reaction. We conclude that, although the suppression of 4-H(P)NE formation from monohydroperoxides by α-tocopherol indicates peroxyl radical reactions in the major route of carbon chain cleavage, the dihydroperoxides previously implicated are not intermediates in the autoxidative transformation of monohydroperoxy fatty acids to 4-HPNE and related aldehydes.

Synthesis of bacillithiol and the catalytic selectivity of FosB-type fosfomycin resistance proteins.

Bacillithiol (BSH) has been prepared on the gram scale from the inexpensive starting material, D-glucosamine hydrochloride, in 11 steps and 8-9% overall yield. The BSH was used to survey the substrate and metal-ion selectivity of FosB enzymes from four Gram-positive microorganisms associated with the deactivation of the antibiotic fosfomycin. The in vitro results indicate that the preferred thiol substrate and metal ion for the FosB from Staphylococcus aureus are BSH and Ni(II), respectively. However, the metal-ion selectivity is less distinct with FosB from Bacillus subtilis, Bacillus anthracis, or Bacillus cereus.

Glucose Autoxidation Induces Functional Damage to Proteins via Modification of Critical Arginine Residues

Nonenzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-α3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to α(V)β(3) integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while nonoxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation.

Inhalable Curcumin: Offering the Potential for Translation to Imaging and Treatment of Alzheimer's Disease

Curcumin is a promising compound that can be used as a theranostic agent to aid research in Alzheimers disease. Beyond its ability to bind to amyloid plaques, the compound can also cross the blood-brain barrier. Presently, curcumin can be applied only to animal models, as the formulation needed for iv injection renders it unfit for human use. Here, we describe a novel technique to aerosolize a curcumin derivative, FMeC1, and facilitate its safe delivery to the brain. Aside from the translational applicability of this approach, a study in the 5XFAD mouse model suggested that inhalation exposure to an aerosolized FMeC1 modestly improved the distribution of the compound in the brain. Additionally, immunohistochemistry data confirms that following aerosol delivery, FMeC1 binds amyloid plaques expressed in the hippocampal areas and cortex.

Structural identification of Diindole agonists of the aryl hydrocarbon receptor derived from degradation of indole-3-pyruvic acid.

Aerobic incubation of the tryptophan transamination/oxidation product indole-3-pyruvic acid (I3P) at pH 7.4 and 37 degrees C yielded products with activity as Ah receptor (AHR) agonists. The extracts were fractionated using HPLC and screened for AHR agonist activity. Two compounds were identified as agonists: 1,3-di(1H-indol-3-yl)propan-2-one (1) and 1-(1H-indol-3-yl)-3-(3H-indol-3-ylidene) propan-2-one (2), with the potency of 2 being 100-fold > 1 [ Nguyen et al. ( 2009 ) Chem. Res. Toxicol. , DOI: 10.1021/tx900043s . ]. Both 1 and 2 showed UV spectra indicative of indole. The molecular formulas were established by high-resolution mass spectrometry (HRMS), and the structures were determined by a combination of NMR methods, including (1)H, natural abundance (13)C, and two-dimensional methods. An intermediate in the oxidation of I3P to 1 is 3-hydroxy-2,4-di(1H-indol-3-yl)butanal (HRMS established the presence of a compound with the formula C(20)H(19)N(2)O(2)). Compound 1 was converted to 2 in air or (faster) with mild oxidants, and 2 could be further oxidized to 1,3-di(3H-indol-3-ylidene)propan-2-one. Determination of the structures allowed estimation of the molar Ah receptor agonist activity of these natural products, similar in potency to known classical AHR inducers.

Isolation and Characterization of Two Geometric Allene Oxide Isomers Synthesized from 9S-Hydroperoxylinoleic Acid by Cytochrome P450 CYP74C3 STEREOCHEMICAL ASSIGNMENT OF NATURAL FATTY ACID ALLENE OXIDES

Background: Allene oxides involved in cyclopentenone biosynthesis are extremely labile and have eluded full stereochemical assignment. Results: We identify a novel Z-allene oxide that unexpectedly rearranges to a cyclopentenone. Conclusion: Other natural allene oxides are assigned the E configuration and can be easily distinguished from the Z-allene oxide by NMR. Significance: Unequivocal determination of the unknown allene oxide configuration is documented and helps elucidate the cyclization chemistry. Specialized cytochromes P450 or catalase-related hemoproteins transform fatty acid hydroperoxides to allene oxides, highly reactive epoxides leading to cyclopentenones and other products. The stereochemistry of the natural allene oxides is incompletely defined, as are the structural features required for their cyclization. We investigated the transformation of 9S-hydroperoxylinoleic acid with the allene oxide synthase CYP74C3, a reported reaction that unexpectedly produces an allene oxide-derived cyclopentenone. Using biphasic reaction conditions at 0 °C, we isolated the initial products and separated two allene oxide isomers by HPLC at −15 °C. One matched previously described allene oxides in its UV spectrum (λmax 236 nm) and NMR spectrum (defining a 9,10-epoxy-octadec-10,12Z-dienoate). The second was a novel stereoisomer (UV λmax 239 nm) with distinctive NMR chemical shifts. Comparison of NOE interactions of the epoxy proton at C9 in the two allene oxides (and the equivalent NOE experiment in 12,13-epoxy allene oxides) allowed assignment at the isomeric C10 epoxy-ene carbon as Z in the new isomer and the E configuration in all previously characterized allene oxides. The novel 10Z isomer spontaneously formed a cis-cyclopentenone at room temperature in hexane. These results explain the origin of the cyclopentenone, provide insights into the mechanisms of allene oxide cyclization, and define the double bond geometry in naturally occurring allene oxides.

Steric and Electrostatic Effects at the C2 Atom Substituent Influence Replication and Miscoding of the DNA Deamination Product Deoxyxanthosine and Analogs by DNA Polymerases

Deoxyinosine (dI) and deoxyxanthosine (dX) are both formed in DNA at appreciable levels in vivo by deamination of deoxyadenosine (dA) and deoxyguanosine (dG), respectively, and can miscode. Structure-activity relationships for dA pairing have been examined extensively using analogs but relatively few studies have probed the roles of the individual hydrogen-bonding atoms of dG in DNA replication. The replicative bacteriophage T7 DNA polymerase/exonuclease and the translesion DNA polymerase Sulfolobus solfataricus pol IV were used as models to discern the mechanisms of miscoding by DNA polymerases. Removal of the 2-amino group from the template dG (i.e., dI) had little impact on the catalytic efficiency of either polymerase, as judged by either steady-state or pre-steady-state kinetic analysis, although the misincorporation frequency was increased by an order of magnitude. dX was highly miscoding with both polymerases, and incorporation of several bases was observed. The addition of an electronegative fluorine atom at the 2-position of dI lowered the oligonucleotide T(m) and strongly inhibited incorporation of dCTP. The addition of bromine or oxygen (dX) at C2 lowered the T(m) further, strongly inhibited both polymerases, and increased the frequency of misincorporation. Linear activity models show the effects of oxygen (dX) and the halogens at C2 on both DNA polymerases as mainly due to a combination of both steric and electrostatic factors, producing a clash with the paired cytosine O2 atom, as opposed to either bulk or perturbation of purine ring electron density alone.