Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).     

Ceramide Synthesis Is Modulated by the Sphingosine Analog FTY720 via a Mixture of Uncompetitive and Noncompetitive Inhibition in an Acyl-CoA Chain Length-de pend ent Manner

FTY720, a sphingosine analog, is in clinical trials as an immunomodulator. The biological effects of FTY720 are believed to occur after its metabolism to FTY720 phosphate. However, very little is known about whether FTY720 can interact with and modulate the activity of other enzymes of sphingolipid metabolism. We examined the ability of FTY720 to modulate de novo ceramide synthesis. In mammals, ceramide is synthesized by a family of six ceramide synthases, each of which utilizes a restricted subset of acyl-CoAs. We show that FTY720 inhibits ceramide synthase activity in vitro by noncompetitive inhibition toward acyl-CoA and uncompetitive inhibition toward sphinganine; surprisingly, the efficacy of inhibition depends on the acyl-CoA chain length. In cultured cells, FTY720 has a more complex effect, with ceramide synthesis inhibited at high (500 nm to 5 μm) but not low (<200 nm) sphinganine concentrations, consistent with FTY720 acting as an uncompetitive inhibitor toward sphinganine. Finally, electrospray ionization-tandem mass spectrometry demonstrated, unexpectedly, elevated levels of ceramide, sphingomyelin, and hexosylceramides after incubation with FTY720. Our data suggest a novel mechanism by which FTY720 might mediate some of its biological effects, which may be of mechanistic significance for understanding its mode of action.FTY720 (2-amino-(2-2-[4-octylphenyl]ethyl)propane 1,3-diol hydrochloride), also known as Fingolimod, is an immunosuppressant drug currently being tested in clinical trials for organ transplantation and autoimmune diseases such as multiple sclerosis (1). FTY720 is a structural analog of sphingosine, a key biosynthetic intermediate in sphingolipid (SL)² metabolism (see Fig. 1). In vivo, FTY720 is rapidly phosphorylated by sphingosine kinase 2 (2, 3) to form FTY720 phosphate (FTY720-P), an analog of sphingosine 1-phosphate (S1P) (see Fig. 1A). FTY720-P binds to S1P receptors (S1PRs) (4, 5) and thereby induces a variety of phenomena such as T-lymphocyte migration from lymphoid organs (6–9); accordingly, FTY720 treatment results in lymphopenia as lymphocytes (especially T-cells) become sequestered inside lymphoid organs (10–12). The ability of FTY720 to sequester lymphocytes has stimulated its use in treatment of allograft rejection and autoimmune diseases (13), and FTY720 is currently under phase III clinical trials for treatment of relapsing-remitting multiple sclerosis (14).Open in a separate windowFIGURE 1.SL structure and metabolism. A, structures of SLs and SL analogs used in this study. B, metabolic inter-relationships between SLs and the metabolism of FTY720. The enzymes are denoted in italics. LPP3, lipid phosphate phosphatase 3; LPP1α, lipid phosphate phosphatase 1α.Apart from the binding of FTY720-P to S1PRs, the ability of FTY720 to inhibit S1P lyase (15) (see Fig. 1B), and its inhibitory effect on cytosolic phospholipase A2 (16), whose activity can be modulated by ceramide 1-phosphate (17), little is known about whether FTY720 or FTY720-P can modulate the activity of other enzymes of SL metabolism. Because FTY720 is an analog of sphingosine, one of the two substrates of ceramide synthase (CerS) (see Fig. 1), we now examine whether FTY720 can modulate CerS activity. CerS utilizes fatty acyl-CoAs to N-acylate sphingoid long chain bases. Six CerS exist in mammals, each of which uses a restricted subset of acyl-CoAs (18–23). We demonstrate that FTY720 inhibits CerS activity and that the extent of inhibition varies according to the acyl chain length of the acyl-CoA substrate. Surprisingly, FTY720 inhibits CerS activity toward acyl-CoA via noncompetitive inhibition and toward sphinganine via uncompetitive inhibition. Finally, the mode of interaction of FTY720 with CerS in cultured cells depends on the amount of available sphinganine. Together, we show that FTY720 modulates ceramide synthesis, which may be of relevance for understanding its biological effects in vivo and its role in immunomodulation.     

Novel Lipofuscin Bisretinoids Prominent in Human Retina and in a Model of Recessive Stargardt Disease

Bisretinoid adducts accumulate as lipofuscin in retinal pigment epithelial (RPE) cells of the eye and are implicated in the pathology of inherited and age-related macular degeneration. Characterization of the bisretinoids A2E and the all-trans-retinal dimer series has shown that these pigments form from reactions in photoreceptor cell outer segments that involve all-trans-retinal, the product of photoisomerization of the visual chromophore 11-cis-retinal. Here we have identified two related but previously unknown RPE lipofuscin compounds. By high performance liquid chromatography-elec tro spray ionization-tandem mass spectrometry, we determined that the first of these compounds is a phosphatidyl-dihydropyridine bisretinoid; to indicate this structure and its formation from two vitamin A-aldehyde (A2), we will refer to it as A2-dihydropyridine-phosphatidyleth a nol amine (A2-DHP-PE). The second pigment, A2-dihydropyridine-eth a nol amine, forms from phosphate hydrolysis of A2-DHP-PE. The structure of A2-DHP-PE was corroborated by Fourier transform infrared spectroscopy, and density functional theory confirmed the presence of a dihydropyridine ring. This lipofuscin pigment is a fluorescent compound with absorbance maxima at ∼490 and 330 nm, and it was identified in human, mouse, and bovine eyes. We found that A2-DHP-PE forms in reaction mixtures of all-trans-retinal and phosphatidyleth a nol amine, and in mouse eyecups we observed an age-related accumulation. As compared with wild-type mice, A2-DHP-PE is more abundant in mice with a null mutation in Abca4 (ATP-binding cassette transporter 4), the gene causative for recessive Stargardt macular degeneration. Efforts to clarify the composition of RPE lipofuscin are important because these compounds are targets of gene-based and drug therapies that aim to alleviate ABCA4-related retinal disease.Throughout the life of an individual, retinal pigment epithelial (RPE)² cells of the eye accumulate bisretinoid adducts that comprise the lipofuscin of these cells (1–3). The compounds form as a byproduct of light-mediated isomerization of the visual chromophore 11-cis-retinal. Accordingly, conditions that reduce the production of all-trans-retinal (atRAL) from 11-cis-retinal photoisomerization, such as reduced serum vitamin A (4–6), variants, or mutations in the visual cycle protein RPE65 (7–9) and inhibitors of RPE65 and 11-cis retinol dehydrogenase (10–13), substantially reduce the formation of this material.Up to the present time, at least 17 constituents of RPE lipofuscin have been identified chromatographically and characterized structurally; added to these are biosynthetic intermediates such as N-retinylidene-phosphatidylethanolamine (NRPE), A2PE and dihydropyridinium-A2PE (see Fig. 1, A and B) (14–19). The first RPE lipofuscin constituent to be described was A2E (see Fig. 1A, inset). The pyridinium bisretinoid (14–16, 20, 21) structure of A2E (C₄₂H₅₈NO; molecular weight, 592) was confirmed by extensive nuclear magnetic resonance studies (14) and by total synthesis (22). A2E formation begins in photoreceptor outer segments when atRAL, instead of being reduced to all-trans-retinol, reacts with phosphatidylethanolamine (PE) in a 2:1 ratio. Although the double bonds along the side arms of A2E are all in the trans (E) position, Z-isomers of A2E have double bonds at the C-13/14 (isoA2E), C-9/9′-10/10′, and C-11/11′-12/12′ positions, and all are detectable in human and mouse RPE (16). These pigments exhibit absorbances in both the UV and visible regions of the spectrum (A2E: λ_max, 439 and 338 nm; iso-A2E: λ_max, 428 and 337 nm).Open in a separate windowFIGURE 1.Some bisretinoid compounds associated with RPE lipofuscin formation. Structures, molecular weight (Mw), UV-visible absorbance (nm), and electronic transition assignments (↔). Phosphate hydrolysis (dashed lines) of A2PE (A) and A2-DHP-PE (C) generates A2E and A2-DHP-E (insets in A and C). A2PE (A), dihydropyridinium-A2PE (B), A2-DHP-PE (C). The molecular weights are based on dipalmitic acid as the fatty acid constituent.Another bisretinoid compound of RPE lipofuscin also absorbs in the short wavelength region of the visible spectrum (17, 18, 23). This pigment, all-trans-retinal dimer (atRAL dimer; λ_max, 432 and 290 nm) forms from the condensation of two atRAL and is present in RPE lipofuscin as Schiff base conjugates with either PE or ethanolamine (atRAL dimer-PE and atRAL dimer-E, respectively) or as unconjugated atRAL dimer. The pigments atRAL dimer-PE and atRAL dimer-E absorb in the visible range at about 510 nm, a “red” shift relative to atRAL dimer that is attributable to protonation of the Schiff base linkage. Although A2E is a pyridinium salt containing a quaternary amine nitrogen that does not deprotonate or reprotonate (24), the protonation state of the Schiff base linkage in atRAL dimer-PE and atRAL dimer-E is pH-dependent (18).Other known constituents of RPE lipofuscin are generated by photooxidation. By mass spectrometry, the photooxidation products of A2E and atRAL dimer present as a series of peaks differing by increments of mass 16 beginning with M+ 592 (A2E) or M+ 552 (atRAL dimer) (18, 25). The moieties generated by the addition of oxygens at CC bonds of these bisretinoid compounds include endoperoxides, furanoid oxides, and epoxides (25–27). These oxidized products are more polar than the parent compound, and mono- and bis-oxidized forms of A2E and atRAL dimer have been detected in RPE from human eyes and in eyecups from mice with null mutations in Abca4^−/− (18, 25), the gene responsible for recessive Stargardt macular degeneration. It is also notable that unconjugated atRAL dimer is a more efficient generator of singlet oxygen than is A2E and is also a more efficient quencher of singlet oxygen (18).Insight into the composition of RPE lipofuscin and the biosynthetic pathways by which these compounds form aids in an understanding of retinal diseases characterized by lipofuscin overload, particularly those associated with mutations in ABCA4 (ATP-binding cassette transporter 4) of photoreceptor cells (1–3). We report that a previously unrecognized bisretinoid molecule absorbing with maxima at 490 and 331 nm is detected at elevated levels in Abca4^−/− mice, a model of recessive Stargardt macular degeneration. This compound is also present in human RPE. By high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS), with corroboration by Fourier transform infrared spectroscopy (FTIR), we determined that this molecule is a bisretinoid presenting with a noncharged dihydropyridine core (Fig. 1C). We propose a biosynthetic pathway by which this pigment may form and demonstrate that enzymatic removal of the phosphatidic acid portion of the molecule generates a second novel component of RPE lipofuscin.     

Genetic Analysis of the nuo Locus,Which Encodes the Proton-Translocating NADH Dehydrogenase in Escherichia coli

Complex I (EC 1.6.99.3) of the bacterium Escherichia coli is considered to be the minimal form of the type I NADH dehydrogenase, the first enzyme complex in the respiratory chain. Because of its small size and relative simplicity, the E. coli enzyme has become a model used to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex. To begin dissecting the processes by which E. coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus, which encodes the 14 Nuo subunits comprising E. coli complex I. Here we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.

Complex I (NADH:ubiquinone oxidoreductase; EC 1.6.99.3), a type I NADH dehydrogenase that couples the oxidation of NADH to the generation of a proton motive force, is the first enzyme complex of the respiratory chain (2, 35, 47). The Escherichia coli enzyme, considered to be the minimal form of complex I, consists of 14 subunits instead of the 40 to 50 subunits associated with the homologous eukaryotic mitochondrial enzyme (17, 29, 30, 48–50). E. coli also possesses a second NADH dehydrogenase, NDH-II, which does not generate a proton motive force (31). E. coli complex I resembles eukaryotic complex I in many ways (16, 17, 30, 49): it performs the same enzymatic reaction and is sensitive to a number of the same inhibitors, it consists of subunits homologous to those found in all proton-translocating NADH:ubiquinone oxidoreductases studied thus far, it is comprised of a large number of subunits relative to the number that comprise other respiratory enzymes, and it contains flavin mononucleotide and FeS center prosthetic groups. Additionally, it possesses an L-shaped topology (14, 22) like that of its Neurospora crassa homolog (27), and it consists of distinct fragments or subcomplexes. Whereas eukaryotic complex I can be dissected into a peripheral arm and a membrane arm, the E. coli enzyme consists of three subcomplexes referred to as the peripheral, connecting, and membrane fragments (29) (Fig. ​(Fig.1A).1A). The subunit composition of these three fragments correlates approximately with the organization of the 14 structural genes (nuoA to nuoN) (49) of the nuo (for NADH:ubiquinone oxidoreductase) locus (Fig. ​(Fig.1B),1B), an organization that is conserved in several other bacteria, including Salmonella typhimurium (3), Paracoccus denitrificans (53), Rhodobacter capsulatus (12), and Thermus thermophilus (54). The 5′ half of the locus contains a promoter (nuoP), previously identified and located upstream of nuoA (8, 49), and the majority of genes that encode subunits homologous to the nucleus-encoded subunits of eukaryotic complex I and to subunits of the Alcaligenes eutrophus NAD-reducing hydrogenase (17, 29, 30, 49). In contrast, the 3′ half contains the majority of the genes that encode subunits homologous to the mitochondrion-encoded subunits of eukaryotic complex I and to subunits of the E. coli formate-hydrogen lyase complex (17, 29, 30, 49). Whereas the nuclear homologs NuoE, NuoF, and NuoG constitute the peripheral fragment (also referred to as the NADH dehydrogenase fragment [NDF]), the nuclear homologs NuoB, NuoC, NuoD, and NuoI constitute the connecting fragment. The mitochondrial homologs NuoA, NuoH, NuoJ, NuoK, NuoL, NuoM, and NuoN constitute the membrane fragment (29). E. coli complex I likely evolved by fusion of preexisting protein assemblies constituting modules for electron transfer and proton translocation (17–19, 30). Open in a separate windowFIG. 1Schematic of E. coli complex I and the nuo locus. Adapted with permission of the publisher (17, 29, 30, 49). (A) E. coli complex I is comprised of three distinct fragments: the peripheral (light gray), connecting (white), and membrane (dark gray) fragments (17, 29). The peripheral fragment (NDF) is comprised of the nuclear homologs NuoE, -F, and -G and exhibits NADH dehydrogenase activity that oxidizes NADH to NAD⁺; the connecting fragment is comprised of the nuclear homologs NuoB, -C, -D, and -I; and the membrane fragment is comprised of the mitochondrial homologs NuoA, -H, and -J to -N and catalyzes ubiquinone (Q) to its reduced form (QH₂). FMN, flavin mononucleotide. (B) The E. coli nuo locus encodes the 14 Nuo subunits that constitute complex I. The 5′ half of the locus contains a previously identified promoter (nuoP) and the majority of genes that encode the peripheral and connecting subunits (light gray and white, respectively). The 3′ half of the locus contains the majority of the genes encoding the membrane subunits (dark gray). The 3′ end of nuoG encodes a C-Terminal region (CTR) of the NuoG subunit (hatched).Because of its smaller size and relative simplicity, researchers recently have begun to utilize complex I of E. coli, and that of its close relative S. typhimurium, to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex (3, 8, 46) and to investigate the diverse physiological consequences caused by defects in this enzyme (4, 6, 10, 40, 59). Such defects affect the ability of cells to perform chemotaxis (40), to grow on certain carbon sources (4, 6, 10, 40, 57), to survive stationary phase (59), to perform energy-dependent proteolysis (4), to regulate the expression of at least one gene (32), and to maintain virulence (5).To begin dissecting the processes by which E. coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus. Here, we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.     

Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

The FAD-dependent choline oxidase has a flavin cofactor covalently attached to the protein via histidine 99 through an 8α-N(3)-histidyl linkage. The enzyme catalyzes the four-electron oxidation of choline to glycine betaine, forming betaine aldehyde as an enzyme-bound intermediate. The variant form of choline oxidase in which the histidine residue has been replaced with asparagine was used to investigate the contribution of the 8α-N(3)-histidyl linkage of FAD to the protein toward the reaction catalyzed by the enzyme. Decreases of 10-fold and 30-fold in the k_cat/K_m and k_cat values were observed as compared with wild-type choline oxidase at pH 10 and 25 °C, with no significant effect on k_cat/K_O using choline as substrate. Both the k_cat/K_m and k_cat values increased with increasing pH to limiting values at high pH consistent with the participation of an unprotonated group in the reductive half-reaction and the overall turnover of the enzyme. The pH independence of both ^D(k_cat/K_m) and ^Dk_cat, with average values of 9.2 ± 3.3 and 7.4 ± 0.5, respectively, is consistent with absence of external forward and reverse commitments to catalysis, and the chemical step of CH bond cleavage being rate-limiting for both the reductive half-reaction and the overall enzyme turnover. The temperature dependence of the ^Dk_red values suggests disruption of the preorganization in the asparagine variant enzyme. Altogether, the data presented in this study are consistent with the FAD-histidyl covalent linkage being important for the optimal positioning of the hydride ion donor and acceptor in the tunneling reaction catalyzed by choline oxidase.A number of enzymes, including dehydrogenases (1–3), monooxygenases (4–7), halogenases (8–11), and oxidases (7, 12, 13), employ flavin cofactors (FAD or FMN) for their catalytic processes. About a tenth of all flavoproteins have been shown to contain a covalently attached cofactor, which may be linked at the C8M position via histidyl, tyrosyl, or cysteinyl side chains or at the C6M position via a cysteinyl side chain (14). Glucooligosaccharide oxidase (15, 16), hexose oxidase (17), and berberine bridge enzyme (18, 19) are examples of flavoproteins (FAD as cofactor) with both linkages present in one flavin molecule. The covalent linkages in flavin-dependent enzymes have been shown to stabilize protein structure (20–22), prevent loss of loosely bound flavin cofactors (23), modulate the redox potential of the flavin microenvironment (20, 23–27), facilitate electron transfer reactions (28), and contribute to substrate binding as in the case of the cysteinyl linkage (20). However, no study has implicated a mechanistic role of the flavin covalent linkages in enzymatic reactions in which a hydride ion is transferred by quantum mechanical tunneling.The discovery of quantum mechanical tunneling in enzymatic reactions, in which hydrogen atoms, protons, and hydride ions are transferred, has attracted considerable interest in enzyme studies geared toward understanding the mechanisms underlying the several orders of magnitudes in the rate enhancements of protein-catalyzed reactions compared with non-enzymatic ones. Tunneling mechanisms have been shown in a wide array of cofactor-dependent enzymes, including flavoenzymes. Examples of flavoenzymes in which the tunneling mechanisms have been demonstrated include morphinone reductase (29, 30), pentaerythritol tetranitrate reductase (29), glucose oxidase (31–33), and choline oxidase (34). Mechanistic data on Class 2 dihydroorotate dehydrogenases, also with a flavin cofactor (FMN) covalently linked to the protein moiety (35, 36), could only propose a mechanism that is either stepwise or concerted with significant quantum mechanical tunneling for the hydride transfer from C6 and the deprotonation at C5 in the oxidation of dihydroorotate to orotate (37). This leaves choline oxidase as the only characterized enzyme with a covalently attached flavin cofactor (12, 38), where the oxidation of its substrate occurs unequivocally by quantum mechanical tunneling.Choline oxidase from Arthrobacter globiformis catalyzes the two-step FAD-dependent oxidation of the primary alcohol substrate choline to glycine betaine with betaine aldehyde, which is predominantly bound to the enzyme and forms a gem-diol species, as intermediate (Scheme 1). Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism against stress conditions, thus making genetic engineering of relevant plants of economic interest (39–45), and the biosynthetic pathway for the osmolyte is a potential drug target in human microbial infections of clinical interest (46–48). The first oxidation step catalyzed by choline oxidase involves the transfer of a hydride ion from a deprotonated choline to the protein-bound flavin followed by reaction of the anionic flavin hydroquinone with molecular oxygen to regenerate the oxidized FAD (for a recent review see Ref. 50). The gem-diol choline, i.e. hydrated betaine aldehyde, is the substrate for the second oxidation step (49), suggesting that the reaction may follow a similar mechanism. The isoalloxazine ring of the flavin cofactor, which is buried within the protein, is physically constrained through a covalent linkage via the C(8) methyl of the flavin and the N(3) atom of the histidine side chain at position 99 (Fig. 1) (12). Also contributing to the physical constrain are the proximity of Ile-103 to the pyrimidine ring and the interactions of the backbone atoms of residues His-99 through Ile-103 with the isoalloxazine ring. The rigid positioning of the isoalloxazine ring could only permit a solvent-excluded cavity of ∼125 ų adjacent to the re face of the FAD to accommodate a 93-ų choline molecule in the substrate binding domain (12). Mechanistic data thus far obtained on choline oxidase, coupled with the crystal structure of the wild-type enzyme resolved to 1.86 Å, are consistent with a quantum tunneling mechanism for the hydride ion transfer occurring within a highly preorganized enzyme-substrate complex (Scheme 2) (12, 34, 50). Exploitation of the tunneling mechanism requires minimal independent movement of the hydride ion donor and acceptor, with the only dynamic motions permitted being the ones that promote the hydride transfer reaction.Open in a separate windowSCHEME 1.Two-step, four-electron oxidation of choline catalyzed by choline oxidase.Open in a separate windowFIGURE 1.x-ray crystal structure of the active site of wild-type choline oxidase resolved to 1.86 Å (PDB 2jbv). Note the significant distortion of the flavin ring at the C(4a) atom, which is due to the presence of a C(4a) adduct (69).Open in a separate windowSCHEME 2.The hydride ion transfer reaction from the α-carbon of the activated choline alkoxide species to the N(5) atom of the isoalloxazine ring of the enzyme-bound flavin in choline oxidase.In the present study, the contribution of the physically constrained flavin isoalloxazine ring to the reaction catalyzed by choline oxidase has been investigated in a variant enzyme in which the histidine residue at position 99 was replaced with an asparagine. The results suggest that, although not being required per se, the covalent linkage in choline oxidase contributes to the hydride tunneling reaction by either preventing independent movement or contributing to the optimal positioning of the flavin acting as hydride ion acceptor with respect to the alkoxide species acting as a donor. However, the covalent linkage is not required for the reaction.     

5-Lipoxygenase-mediated Endogenous DNA Damage

Lipoxygenases (LOs) convert polyunsaturated fatty acids into lipid hydroperoxides. Homolytic decomposition of lipid hydroperoxides gives rise to endogenous genotoxins such as 4-oxo-2(E)-nonenal, which cause the formation of mutagenic DNA adducts. Chiral lipidomics analysis was employed to show that a 5-LO-derived lipid hydroperoxide was responsible for endogenous DNA-adduct formation. The study employed human lymphoblastoid CESS cells, which expressed both 5-LO and the required 5-LO-activating protein (FLAP). The major lipid peroxidation product was 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid, which was analyzed as its reduction product, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5(S)-HETE)). Concentrations of 5(S)-HETE increased from 0.07 ± 0.01 to 45.50 ± 4.05 pmol/10⁷ cells upon stimulation of the CESS cells with calcium ionophore A23187. There was a concomitant increase in the 4-oxo-2(E)-nonenal-derived DNA-adduct, heptanone-etheno-2′-deoxyguanosine (HϵdGuo) from 2.41 ± 0.35 to 6.31 ± 0.73 adducts/10⁷ normal bases. Biosynthesis of prostaglandins, 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid, and 15(R,S)-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid revealed that there was cyclooxygenase (COX) activity in the CESS cells. Western blot analysis revealed that COX-1 was expressed by the cells, but there was no COX-2 or 15-LO-1. FLAP inhibitor reduced HϵdGuo-adducts and 5(S)-HETE to basal levels. In contrast, aspirin, which had no effect on 5(S)-HETE, blocked the formation of prostaglandins, 15-HETE, and 11-HETE but did not inhibit HϵdGuo-adduct formation. These data showed that 5-LO was the enzyme responsible for the generation of the HϵdGuo DNA-adduct in CESS cells.PUFAs² can be converted into lipid hydroperoxides enzymatically by the action of LOs (1) and COXs (2) or nonenzymatically by reactive oxygen species (ROS) (3). Arachidonic acid, one of the essential PUFAs present in cell membranes, is released from phospholipids by different phospholipase A₂ isoforms upon diverse physical, chemical, inflammatory, and mitogenic stimuli (4). The free arachidonic acid then serves as a substrate for LOs, COXs, or ROS to produce a variety of lipid hydroperoxides (5, 6). ROS, 12-LO, and 15-LO can also act directly upon arachidonic acid esterified in phospholipids to produce lipid hydroperoxides (7), which are reduced (8), hydrolyzed by phospholipase A₂ (4), and then secreted as the corresponding free HETEs (9). COX-2-mediated (10) and 15-LO-1-mediated (11) metabolism of linoleic acid results in the formation of 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, which is rapidly reduced to 13(S)-hydroxy-9,11-(Z,E)-octadecadienoic acid (HODE) and secreted from cells. Arachidonic acid is specifically metabolized by 5-LO into 5(S)-HpETE, which is either reduced to 5(S)-HETE or serves as precursor to the formation of leukotrienes (LTs) (Scheme 1) (12). In contrast, ROS-mediated reactions produce racemic mixtures of all possible regioisomers of HpETEs and 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acids (3) that are subsequently secreted from cells as complex mixtures of racemic HETEs and HODEs. Therefore, the ability to analyze different HETE and HODE enantiomers and regioisomers is important for elucidating specific cellular lipid peroxidation pathways (13).Open in a separate windowSCHEME 1.5-LO-mediated formation of arachidonic acid metabolites and dGuo-adducts. HPNE, 4-hydroperoxy-2(E)-nonenal; DOOE, dioxo-6-octenoic acid.The conversion of arachidonic acid to 5(S)-HpETE by 5-LO is critically dependent upon the presence of FLAP (14). 5-LO and FLAP are expressed primarily in inflammatory cells such as polymorphonuclear leukocytes, monocytes, macrophages, and mast cells (12, 15–17). Therefore, 5-LO-mediated LT formation is thought to play a critical role in inflammation and allergic disorders (18–21). In addition, a number of studies have implicated 5-LO-derived arachidonic acid metabolites as mediators of atherogenesis and heart disease (12, 22, 23). The 5-LO pathway of arachidonic acid metabolism has also been proposed to play a role in prostate and pancreatic cancer (24–26).Lipid hydroperoxides undergo homolytic decomposition into bifunctional electrophiles such as 4-hydroxy-2(E)-nonenal, ONE, 4,5-epoxy-2(E)-decenal, and 4-hydroperoxy-2(E)-nonenal (27). These bifunctional electrophiles are highly reactive and can readily modify intracellular molecules including glutathione (GSH) (28, 29), DNA, (5, 6), and proteins (30, 31). Our previous in vitro studies characterized the bifunctional electrophiles ONE and 4-hydroperoxy-2(E)-nonenal as major products arising from the homolytic decomposition of 5-LO-derived 5(S)-HpETE (32). Reactions with DNA resulted in the formation of etheno-2′-deoxyguanosine (ϵdGuo) from 4-hydroperoxy-2(E)-nonenal and heptanone-ϵdGuo (HϵdGuo) from ONE (Scheme 1). 5,8-Dioxo-6-octenoic acid, a bifunctional electrophile from the carboxyl terminus of 5(S)-HpETE, gave rise to the novel DNA-adduct carboxypentanone-ϵdGuo (CPϵdGuo)-adduct as shown in Scheme 1.Previous studies have demonstrated that lipid hydroperoxides generated by COX-2 could lead to the formation of endogenous DNA adducts in epithelial cells (6). Cellular 5-LO, like COX-2, synthesizes lipid hydroperoxides on the nuclear membrane. Therefore, it is highly possible that 5-LO could also mediate the formation of lipid hydroperoxide-derived endogenous DNA adducts in cells. CESS is a human lymphoblastic cell line that expresses both 5-LO and FLAP, and they have been used as a model for inflammatory cells to examine the role of 5-LO metabolites in signal transduction (33, 34). In the present study, CESS cells provided an ideal model to elucidate the relationship of 5-LO mediated-lipid peroxidation and DNA-adduct formation in a cellular setting. Stable isotope dilution chiral LC-electron capture (EC) APCI/MRM/MS (13) was used to monitor the concomitant formation of lipid hydroperoxides in the presence of different enzyme stimulator or inhibitors. DNA-adduct formation in the same cells was measured by a stable isotope dilution APCI/MRM/MS method. The powerful tool of chiral lipid analysis enabled us to dissect the complicated lipid peroxidation pathways and to correlate them with endogenous DNA-adduct formation. The results demonstrated that 5-LO-mediated lipid peroxidation could cause HϵdGuo formation in cells. This novel finding provided additional explanation for the previous observation that increased 5-LO activity was associated with cancers and cardiovascular diseases (24–26).     

Comparative Studies of Class IIa Bacteriocins of Lactic Acid Bacteria

Four class IIa bacteriocins (pediocin PA-1, enterocin A, sakacin P, and curvacin A) were purified to homogeneity and tested for activity toward a variety of indicator strains. Pediocin PA-1 and enterocin A inhibited more strains and had generally lower MICs than sakacin P and curvacin A. The antagonistic activity of pediocin-PA1 and enterocin A was much more sensitive to reduction of disulfide bonds than the antagonistic activity of sakacin P and curvacin A, suggesting that an extra disulfide bond that is present in the former two may contribute to their high levels of activity. The food pathogen Listeria monocytogenes was among the most sensitive indicator strains for all four bacteriocins. Enterocin A was most effective in inhibiting Listeria, having MICs in the range of 0.1 to 1 ng/ml. Sakacin P had the interesting property of being very active toward Listeria but not having concomitant high levels of activity toward lactic acid bacteria. Strains producing class IIa bacteriocins displayed various degrees of resistance toward noncognate class IIa bacteriocins; for the sakacin P producer, it was shown that this resistance is correlated with the expression of immunity genes. It is hypothesized that variation in the presence and/or expression of such immunity genes accounts in part for the remarkably large variation in bacteriocin sensitivity displayed by lactic acid bacteria.Many lactic acid bacteria (LAB), including members of the genera Lactococcus, Lactobacillus, Carnobacterium, Enterococcus, and Pediococcus, are known to secrete small, ribosomally synthesized antimicrobial peptides called bacteriocins (26, 29, 34). Some of these peptides undergo posttranslational modifications (class I bacteriocins), whereas others are not modified (class II bacteriocins) (29, 34). Class II bacteriocins contain between 30 and 60 residues and are usually positively charged at a neutral pH. Studies of a large number of class II bacteriocins have led to subgrouping of these compounds (29, 34). One of the subgroups, class IIa, contains bacteriocins that are characterized by the presence of YGNG and CXXXXCXV sequence motifs in their N-terminal halves as well as by their strong inhibitory effect on Listeria (e.g., 3, 4, 22, 23, 27, 28, 31, 38, 45) (Fig. ​(Fig.1).1). Because of their effectiveness against the food pathogen Listeria, class IIa bacteriocins have potential as antimicrobial agents in food and feed. Open in a separate windowFIG. 1Sequence alignment of class IIa bacteriocins. Residue numbering is according to the sequence of pediocin PA-1. Cysteine residues are printed in boldface; the two known class IIa bacteriocins with four cysteine residues are in the upper group. No attempt was made to optimize the alignment in the C-terminal halves of the peptides. Piscicolin 126 is identical to piscicocin V1a (4). Carnobacteriocin BM1 most probably is identical to piscicocin V1b (4). Sakacin P most probably is identical to bavaricin A (30). Curvacin A is identical to sakacin A (2). The consensus sequence includes residues conserved in at least 8 of the 12 sequences shown; 100% conserved residues are underlined.Class IIa bacteriocins act by permeabilizing the membrane of their target cells (1, 5, 6, 9, 10, 26, 28). The most recent studies on the mode of action of these bacteriocins indicate that antimicrobial activity does not require a specific receptor and is enhanced by (but not fully dependent on) a membrane potential (9, 28). Little is known about bacteriocin structure, and unravelling the relationships between structure and function is one of the great challenges in current bacteriocin research. A logical starting point for structure-function studies is a thorough study of the differences in activity and target cell specificity between naturally occurring homologous bacteriocins. A few such studies have been described, but these suffer from either a very limited number of tested indicator strains or the use of culture supernatants instead of purified bacteriocins (3, 4, 17, 45). The use of purified bacteriocins for comparative analyses is absolutely essential, since it is becoming increasingly evident that bacteriocin producers produce more than one bacteriocin (4, 8, 38, 48; this study).In the present study, the activities of four pure class IIa bacteriocins (pediocin PA-1, enterocin A, curvacin A, and sakacin P) (Fig. ​(Fig.1)1) were tested against a large number of LAB as well as several strains of the food pathogen Listeria monocytogenes. The bacteriocins were purified from their respective producer strains by use of an optimized purification protocol yielding highly pure samples. The contribution of disulfide formation was assessed and found to be important for activity. The effects of the purified bacteriocins on (noncognate) class IIa bacteriocin-producing strains are described, and the implications of our findings for immunity and resistance are discussed.     

Expression of a Soybean Gene Encoding the Tetrapyrrole-Synthesis Enzyme Glutamyl-tRNA Reductase in Symbiotic Root Nodules

Heme and chlorophyll accumulate to high levels in legume root nodules and in photosynthetic tissues, respectively, and they are both derived from the universal tetrapyrrole precursor δ-aminolevulinic acid (ALA). The first committed step in ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase (GTR) in plants. A soybean (Glycine max) root-nodule cDNA encoding GTR was isolated by complementation of an Escherichia coli GTR-defective mutant for restoration of ALA prototrophy. Gtr mRNA was very low in uninfected roots but accumulated to high levels in root nodules. The induction of Gtr mRNA in developing nodules was subsequent to that of the gene Enod2 (early nodule) and coincided with leghemoglobin mRNA accumulation. Genomic analysis revealed two Gtr genes, Gtr1 and a 3′ portion of Gtr2, which were isolated from the soybean genome. RNase-protection analysis using probes specific to Gtr1 and Gtr2 showed that both genes were expressed, but Gtr1 mRNA accumulated to significantly higher levels. In addition, the qualitative patterns of expression of Gtr1 and Gtr2 were similar to each other and to total Gtr mRNA in leaves and nodules of mature plants and etiolated plantlets. The data indicate that Gtr1 is universal for tetrapyrrole synthesis and that a Gtr gene specific for a tissue or tetrapyrrole is unlikely. We suggest that ALA synthesis in specialized root nodules involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants.Soybean (Glycine max) and numerous other legumes can establish a symbiosis with rhizobia, resulting in the formation of root nodules comprising specialized plant and bacterial cells (for review, see Mylona et al., 1995). Rhizobia reduce atmospheric nitrogen to ammonia within nodules, which is assimilated by the plant host to fulfill its nutritional nitrogen requirement. The high energy requirement for nitrogen fixation necessitates efficient respiration by the prokaryote within the microaerobic milieu of the nodule. The plant host synthesizes a nodule-specific hemoglobin (leghemoglobin) that serves to facilitate oxygen diffusion to the bacterial endosymbiont and to buffer the free oxygen concentration at a low tension (for review, see Appleby, 1992). Both of these functions require that the hemoglobin concentration be high, and, indeed, it exceeds 1 mm in soybean nodules (Appleby, 1984) and is the predominant plant protein in that organ. Once thought to be confined to legume nodules, hemoglobins are found throughout the plant kingdom, and leghemoglobin likely represents a specialization of a general plant phenomenon (for review, see Hardison, 1996). A gene encoding a nonsymbiotic hemoglobin has been identified in soybean and other legumes (Andersson et al., 1996); therefore, expression in nodules involves the specific activation of a subset of genes within a gene family. Leghemoglobin genes may have arisen from gene duplication, followed by specialization (Andersson et al., 1996).Hemes and chlorophyll are tetrapyrroles synthesized from common precursors; chlorophyll is quantitatively the major tetrapyrrole in plants, with heme and other tetrapyrroles being present in minor amounts. Legume root nodules represent an exception, in which heme is synthesized in high quantity in the absence of chlorophyll, thus requiring the activity of enzymes not normally expressed highly in nonphotosynthetic tissues. Heme is synthesized from the universal tetrapyrrole precursor ALA by seven successive enzymatic steps; chlorophyll formation diverges after the synthesis of protoporphyrin, the immediate heme precursor (for review, see O''Brian, 1996). Biochemical and genetic evidence shows that soybean heme biosynthesis genes are strongly induced in root nodules (Sangwan and O''Brian, 1991, 1992, 1993; Madsen et al., 1993; Kaczor et al., 1994; Frustaci et al., 1995; Santana et al., 1998), and immunohistochemical studies demonstrate that induction is concentrated in infected nodule cells (Santana et al., 1998).ALA is synthesized from Glu in plants by a three-step mechanism called the C₅ pathway (Fig. ​(Fig.1);1); the latter two steps are committed to ALA synthesis and are catalyzed by GTR and GSAT, respectively (for review, see Beale and Weinstein, 1990; Jahn et al., 1991). Plant cDNA or genes encoding GTR (Gtr, also called HemA) and GSAT (Gsa) have been identified in several plant species (Grimm, 1990; Sangwan and O''Brian, 1993; Hofgen et al., 1994; Ilag et al., 1994; Frustaci et al., 1995; Wenzlau and Berry-Lowe, 1995; Bougri and Grimm, 1996; Kumar et al., 1996; Tanaka et al., 1996). Two genes for each enzyme have been described, and some genes are reported to be specific to a tissue, tetrapyrrole, or light regimen (Bougri and Grimm, 1996; Kumar et al., 1996; Tanaka et al., 1996). However, soybean Gsa1 is highly expressed in both leaves and nodules and contains a cis-acting element in its promoter that binds to a nuclear factor found in both tissues. (Frustaci et al., 1995). In this study we isolated soybean Gtr1 and characterized the genetic basis of GTR expression in root nodules. Figure 1C₅ pathway for ALA synthesis. The committed steps for ALA synthesis catalyzed by GTR and GSAT are boxed. Glutamyl-tRNA synthetase (GluRS) and glutamyl-tRNA^Glu also participate in protein synthesis. The gene designations in plants are shown in parentheses ...