Redox regulation of UDP-glucose pyrophosphorylase from Entamoeba histolytica

Amoebiasis is an intestinal infection caused by the human pathogen Entamoeba histolytica and representing the third leading cause of death by parasites in the world. Host-parasite interactions mainly involve anchored glycoconjugates localized in the surface of the parasitic cell. In protozoa, synthesis of structural oligo- and polysaccharides occurs via UDP-glucose, generated in a reaction catalyzed by UDP-glucose pyrophosphorylase. We report the molecular cloning of the gene coding for this enzyme from genomic DNA of E. histolytica and its recombinant expression in Escherichia coli cells. The purified enzyme was kinetically characterized, catalyzing UDP-glucose synthesis and pyrophosphorolysis with V_max values of 95 U/mg and 3 U/mg, respectively, and affinity for substrates comparable to those found for the enzyme from other sources. Enzyme activity was affected by redox modification of thiol groups. Different oxidants, including diamide, hydrogen peroxide and sodium nitroprusside inactivated the enzyme. The process was completely reverted by reducing agents, mainly cysteine, dithiothreitol, and thioredoxin. Characterization of the enzyme mutants C94S, C108S, C191S, C354S, C378S, C108/378S, M106S and M106C supported a molecular mechanism for the redox regulation. Molecular modeling confirmed the role of specific cysteine and methionine residues as targets for redox modification in the entamoebic enzyme. Our results suggest that UDP-glucose pyrophosphorylase is a regulated enzyme in E. histolytica. Interestingly, results strongly agree with the occurrence of a physiological redox mechanism modulating enzyme activity, which would critically affect carbohydrate metabolism in the protozoon.     

Purification and Molecular Characterization of the Tungsten-Containing Formaldehyde Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon Pyrococcus furiosus: the Third of a Putative Five-Member Tungstoenzyme Family

Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100°C by fermenting peptides and sugars to produce organic acids, CO₂, and H₂. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS⁻) under reducing conditions. FOR uses P. furiosus ferredoxin as an electron acceptor (K_m = 100 μM) and oxidizes a range of aldehydes. Formaldehyde (K_m = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C₅ semi- or dialdehyde, e.g., glutaric dialdehyde (K_m = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosus chromosome. Two additional (and nonlinked) genes (termed wor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.     

Redox chemistry of biological tungsten: an EPR study of the aldehyde oxidoreductase from Pyrococcus furiosus

 Aldehyde:ferredoxin oxidoreductase (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus is a homodimeric protein. Each subunit carries one [4Fe-4S] cubane and a novel tungsten cofactor containing two pterins. A single iron atom bridges between the subunits. AOR has previously been studied with EPR spectroscopy in an inactive form known as the red tungsten protein (RTP): reduced RTP exhibits complex EPR interaction signals. We have now investigated the active enzyme AOR with EPR, and we have found an S = 1/2 plus S = 3/2 spin mixture from a non-interacting [4Fe-4S]¹⁺ cluster in the reduced enzyme. Oxidized AOR affords EPR signals typical for W(V) with g–values of 1.982, 1.953, and 1.885. The W(V) signals disappear at a reduction potential E _m,7.5 of +180 mV. This unexpectedly high value indicates that the active-site redox chemistry is based on the pterin part of the cofactor. Received: 18 December 1995 / Accepted: 26 March 1996     

Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii

Methods for efficient growth and manipulation of relatively uncharacterized bacteria facilitate their study and are essential for genetic manipulation. We report new growth media and culture techniques for Caldicellulosiruptor bescii, the most thermophilic cellulolytic bacterium known. A low osmolarity defined growth medium (LOD) was developed that avoids problems associated with precipitates that form in previously reported media allowing the monitoring of culture density by optical density at 680 nm (OD₆₈₀) and more efficient DNA transformation by electroporation. This is a defined minimal medium and does not support growth when a carbon source is omitted, making it suitable for selection of nutritional markers as well as the study of biomass utilization by C. bescii. A low osmolarity complex growth medium (LOC) was developed that dramatically improves growth and culture viability during storage, making it a better medium for routine growth and passaging of C. bescii. Both media contain significantly lower solute concentration than previously published media, allowing for flexibility in developing more specialized media types while avoiding the issues of growth inhibition and cell lysis due to osmotic stress. Plating on LOD medium solidified by agar results in ~1,000-fold greater plating efficiency than previously reported and allows the isolation of discrete colonies. These new media represent a significant advance for both genetic manipulation and the study of biomass utilization in C. bescii, and may be applied broadly across the Caldicellulosiruptor genus.     

Degree of C(4) Photosynthesis in C(4) and C(3)-C(4)Flaveria Species and Their Hybrids : I. CO(2) Assimilation and Metabolism and Activities of Phosphoenolpyruvate Carboxylase and NADP-Malic Enzyme

The degree of C₄ photosynthesis was assessed in four hybrids among C₄, C₄-like, and C₃-C₄ species in the genus Flaveria using ¹⁴C labeling, CO₂ exchange, ¹³C discrimination, and C₄ enzyme activities. The hybrids incorporated from 57 to 88% of the ¹⁴C assimilated in a 10-s exposure into C₄ acids compared with 26% for the C₃-C₄ species Flaveria linearis, 91% for the C₄ species Flaveria trinervia, and 87% for the C₄-like Flaveria brownii. Those plants with high percentages of ¹⁴C initially fixed into C₄ acids also metabolized the C₄ acids quickly, and the percentage of ¹⁴C in 3-phosphoglyceric acid plus sugar phosphates increased for at least a 30-s exposure to ¹²CO₂. This indicated a high degree of coordination between the carbon accumulation and reduction phases of the C₄ and C₃ cycles. Synthesis and metabolism of C₄ acids by the species and their hybrids were highly and linearly correlated with discrimination against ¹³C. The relationship of ¹³C discrimination or ¹⁴C metabolism to O₂ inhibition of photosynthesis was curvilinear, changing more rapidly at C₄-like values of ¹⁴C metabolism and ¹³C discrimination. Incorporation of initial ¹⁴C into C₄ acids showed a biphasic increase with increased activities of phosphoenolpyruvate carboxylase and NADP-malic enzyme (steep at low activities), but turnover of C₄ acids was linearly related to NADP-malic enzyme activity. Several other traits were closely related to the in vitro activity of NADP-malic enzyme but not phosphoenolpyruvate carboxylase. The data indicate that the hybrids have variable degrees of C₄ photosynthesis but that the carbon accumulation and reduction portions of the C₄ and C₃ cycles are well coordinated.     

Simultaneous Involvement of a Tungsten-Containing Aldehyde:Ferredoxin Oxidoreductase and a Phenylacetaldehyde Dehydrogenase in Anaerobic Phenylalanine Metabolism

Anaerobic phenylalanine metabolism in the denitrifying betaproteobacterium Aromatoleum aromaticum is initiated by conversion of phenylalanine to phenylacetate, which is further metabolized via benzoyl-coenzyme A (CoA). The formation of phenylacetate is catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, and a phenylacetaldehyde-oxidizing enzyme. The presence of these enzymes was detected in extracts of cells grown with phenylalanine and nitrate. We found that two distinct enzymes are involved in the oxidation of phenylacetaldehyde to phenylacetate, an aldehyde:ferredoxin oxidoreductase (AOR) and a phenylacetaldehyde dehydrogenase (PDH). Based on sequence comparison, growth studies with various tungstate concentrations, and metal analysis of the enriched enzyme, AOR was shown to be a tungsten-containing enzyme, necessitating specific cofactor biosynthetic pathways for molybdenum- and tungsten-dependent enzymes simultaneously. We predict from the genome sequence that most enzymes of molybdopterin biosynthesis are shared, while the molybdate/tungstate uptake systems are duplicated and specialized paralogs of the sulfur-inserting MoaD and the metal-inserting MoeA proteins seem to be involved in dedicating biosynthesis toward molybdenum or tungsten cofactors. We also characterized PDH biochemically and identified both NAD⁺ and NADP⁺ as electron acceptors. We identified the gene coding for the enzyme and purified a recombinant Strep-tagged PDH variant. The homotetrameric enzyme is highly specific for phenylacetaldehyde, has cooperative kinetics toward the substrate, and shows considerable substrate inhibition. Our data suggest that A. aromaticum utilizes PDH as the primary enzyme during anaerobic phenylalanine degradation, whereas AOR is not essential for the metabolic pathway. We hypothesize a function as a detoxifying enzyme if high aldehyde concentrations accumulate in the cytoplasm, which would lead to substrate inhibition of PDH.     

Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii

Caldicellulosiruptor bescii efficiently degrades cellulose, xylan, and native grasses at high temperatures above 70°C under anaerobic conditions. C. bescii extracellularly secretes multidomain glycoside hydrolases along with proteins of unknown function. In this study, we analyzed the C. bescii proteins that bind to the cell walls of timothy grass by using mass spectrometry, and we identified four noncatalytic plant cell wall-binding proteins (PWBPs) with high pI values (9.2 to 9.6). A search of a conserved domain database showed that these proteins possess a common domain related to solute-binding proteins. In addition, 12 genes encoding PWBP-like proteins were detected in the C. bescii genomic sequence. To analyze the binding properties of PWBPs, recombinant PWBP57 and PWBP65, expressed in Escherichia coli, were prepared. The PWBPs displayed a wide range of binding specificities: they bound to cellulose, lichenan, xylan, arabinoxylan, glucuronoxylan, mannan, glucomannan, pectin, oligosaccharides, and the cell walls of timothy grass. The proteins showed the highest binding affinity for the plant cell wall, with association constant (K_a) values of 5.2 × 10⁶ to 44 × 10⁶ M⁻¹ among the insoluble polysaccharides tested, as measured using depletion binding isotherms. Affinity gel electrophoresis demonstrated that the proteins bound to the acidic polymer pectin most strongly among the soluble polysaccharides tested. Fluorescence microscopic analysis showed that the proteins bound preferentially to the cell wall in a section of grass leaf. Binding of noncatalytic PWBPs with high pI values might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures.     

Xanthine Oxidoreductase Function Contributes to Normal Wound Healing

Chronic, nonhealing wounds result in patient morbidity and disability. Reactive oxygen species (ROS) and nitric oxide (NO) are both required for normal wound repair, and derangements of these result in impaired healing. Xanthine oxidoreductase (XOR) has the unique capacity to produce both ROS and NO. We hypothesize that XOR contributes to normal wound healing. Cutaneous wounds were created in C57Bl6 mice. XOR was inhibited with dietary tungsten or allopurinol. Topical hydrogen peroxide (H₂O₂, 0.15%) or allopurinol (30 μg) was applied to wounds every other day. Wounds were monitored until closure or collected at d 5 to assess XOR expression and activity, cell proliferation and histology. The effects of XOR, nitrite, H₂O₂ and allopurinol on keratinocyte cell (KC) and endothelial cell (EC) behavior were assessed. We identified XOR expression and activity in the skin and wound edges as well as granulation tissue. Cultured human KCs also expressed XOR. Tungsten significantly inhibited XOR activity and impaired healing with reduced ROS production with reduced angiogenesis and KC proliferation. The expression and activity of other tungsten-sensitive enzymes were minimal in the wound tissues. Oral allopurinol did not reduce XOR activity or alter wound healing but topical allopurinol significantly reduced XOR activity and delayed healing. Topical H₂O₂ restored wound healing in tungsten-fed mice. In vitro, nitrite and H₂O₂ both stimulated KC and EC proliferation and EC migration. These studies demonstrate for the first time that XOR is abundant in wounds and participates in normal wound healing through effects on ROS production.     

Homologous Expression of the Caldicellulosiruptor bescii CelA Reveals that the Extracellular Protein Is Glycosylated

Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its role in the structure and function of this important enzyme in biomass deconstruction.     

Reconstitution of a Thermostable Xylan-Degrading Enzyme Mixture from the Bacterium Caldicellulosiruptor bescii

Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars. However, the components of xylan-degrading enzymes are often individually characterized, leading to a dearth of research that analyzes synergistic actions of the components of xylan-degrading enzymes. In the present report, six genes predicted to encode components of the xylan-degrading enzymes of the thermophilic bacterium Caldicellulosiruptor bescii were expressed in Escherichia coli, and the recombinant proteins were investigated as individual enzymes and also as a xylan-degrading enzyme cocktail. Most of the component enzymes of the xylan-degrading enzyme mixture had similar optimal pH (5.5 to ∼6.5) and temperature (75 to ∼90°C), and this facilitated their investigation as an enzyme cocktail for deconstruction of xylans. The core enzymes (two endoxylanases and a β-xylosidase) exhibited high turnover numbers during catalysis, with the two endoxylanases yielding estimated k_cat values of ∼8,000 and ∼4,500 s⁻¹, respectively, on soluble wheat arabinoxylan. Addition of side chain-cleaving enzymes to the core enzymes increased depolymerization of a more complex model substrate, oat spelt xylan. The C. bescii xylan-degrading enzyme mixture effectively hydrolyzes xylan at 65 to 80°C and can serve as a basal mixture for deconstruction of xylans in bioenergy feedstock at high temperatures.     

Redox control of oxidative stress responses in the C3–CAM intermediate plant Mesembryanthemum crystallinum

Crassulacean acid metabolism (CAM) is named after the Crassulaceae family of succulent plants, in which this type of metabolism was first discovered at the beginning of the 19th century. In recent years, Mesembryanthemum crystallinum, a facultative halophyte and C₃–CAM intermediate plant, has become a favoured plant for studying stress response mechanisms during C₃–CAM shifts. Recent studies in this and related areas can provide a new model of how such mechanisms could operate for acclimation to high salinity or excess excitation energy. These include roles for photosynthetic electron transport chain components and reactive oxygen species. The diurnal rhythms of catalase, superoxide dismutase and some CAM-related enzyme activities are discussed in relation to the protective role of photorespiration during C₃–CAM transition. The role of excess excitation energy and redox events in the proximity of photosystem II (PSII) in regulation of ascorbate peroxidase (APX), superoxide dismutase (SOD): copper/zinc SOD (Cu/ZnSOD), iron SOD (FeSOD), and NAD(P)-malic enzyme gene expression are also discussed. We suggest a model in which the chloroplast plays a major role in regulation of acclimation to high salinity and/or excess exitation energy.     

Heterologous expression of a β-<Emphasis Type="SmallCaps">d</Emphasis>-glucosidase in <Emphasis Type="Italic">Caldicellulosiruptor bescii</Emphasis> has a surprisingly modest effect on the activity of the exoproteome and growth on crystalline cellulose

Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria so far described and are capable of efficiently utilizing complex lignocellulosic biomass without conventional pretreatment. Previous studies have shown that accumulation of high concentrations of cellobiose and, to a lesser extent, cellotriose, inhibits cellulase activity both in vivo and in vitro and high concentrations of cellobiose are present in C. bescii fermentations after 90 h of incubation. For some cellulolytic microorganisms, β-d-glucosidase is essential for the efficient utilization of cellobiose as a carbon source and is an essential enzyme in commercial preparations for efficient deconstruction of plant biomass. In spite of its ability to grow efficiently on crystalline cellulose, no extracellular β-d-glucosidase or its GH1 catalytic domain could be identified in the C. bescii genome. To investigate whether the addition of a secreted β-d-glucosidase would improve growth and cellulose utilization by C. bescii, we cloned and expressed a thermostable β-d-glucosidase from Acidothermus cellulolyticus (Acel_0133) in C. bescii using the CelA signal sequence for protein export. The effect of this addition was modest, suggesting that β-d-glucosidase is not rate limiting for cellulose deconstruction and utilization by C. bescii.     

Purification and characterization of the tungsten enzyme aldehyde:ferredoxin oxidoreductase from the hyperthermophilic denitrifier Pyrobaculum aerophilum

A tungsten-containing aldehyde:ferredoxin oxidoreductase (AOR) has been purified to homogeneity from Pyrobaculum aerophilum. The N-terminal sequence of the isolated enzyme matches a single open reading frame in the genome. Metal analysis and electron paramagnetic resonance (EPR) spectroscopy indicate that the P. aerophilum AOR contains one tungsten center and one [4Fe-4S]^2+/1+ cluster per 68-kDa monomer. Native AOR is a homodimer. EPR spectroscopy of the purified enzyme that has been reduced with the substrate crotonaldehyde revealed a W(V) species with g_zyx values of 1.952, 1.918, 1.872. The substrate-reduced AOR also contains a [4Fe-4S]¹⁺ cluster with S=3/2 and zero field splitting parameters D=7.5 cm^–1 and E/D=0.22. Molybdenum was absent from the enzyme preparation. The P. aerophilum AOR lacks the amino acid sequence motif indicative for binding of mononuclear iron that is typically found in other AORs. Furthermore, the P. aerophilum AOR utilizes a 7Fe ferredoxin as the putative physiological redox partner, instead of a 4Fe ferredoxin as in Pyrococcus furiosus. This 7Fe ferredoxin has been purified from P. aerophilum, and the amino acid sequence has been identified using mass spectrometry. Direct electrochemistry of the ferredoxin showed two one-electron transitions, at –306 and –445 mV. In the presence of 55 M ferredoxin the AOR activity is 17% of the activity obtained with 1 mM benzyl viologen as an electron acceptor.     

The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

The genome of the thermophilic bacterium Caldicellulosiruptor bescii encodes three multimodular enzymes with identical C-terminal domain organizations containing two consecutive CBM3b modules and one glycoside hydrolase (GH) family 48 (GH48) catalytic module. However, the three proteins differ much in their N termini. Among these proteins, CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) with a GH9/CBM3c binary partner in the N terminus has been shown to use a novel strategy to degrade crystalline cellulose, which leads to its outstanding cellulose-cleaving activity. Here we show that C. bescii Xyn10C (CbXyn10C), the N-terminal GH10 domain from CbXyn10C/Cel48B, can also degrade crystalline cellulose, in addition to heterogeneous xylans and barley β-glucan. The data from substrate competition assays, mutational studies, molecular modeling, and docking point analyses point to the existence of only one catalytic center in the bifunctional xylanase/β-glucanase. The specific activities of the recombinant CbXyn10C on Avicel and filter paper were comparable to those of GH9/CBM3c of the robust CelA expressed in Escherichia coli. Appending one or two cellulose-binding CBM3bs enhanced the activities of CbXyn10C in degrading crystalline celluloses, which were again comparable to those of the GH9/CBM3c-CBM3b-CBM3b truncation mutant of CelA. Since CbXyn10C/Cel48B and CelA have similar domain organizations and high sequence homology, the endocellulase activity observed in CbXyn10C leads us to speculate that CbXyn10C/Cel48B may use the same strategy that CelA uses to hydrolyze crystalline cellulose, thus helping the excellent crystalline cellulose degrader C. bescii acquire energy from the environment. In addition, we also demonstrate that CbXyn10C may be an interesting candidate enzyme for biotechnology due to its versatility in hydrolyzing multiple substrates with different glycosidic linkages.     

Photosynthetic Enzyme Activities and Localization in Mollugo verticillata Populations Differing in the Levels of C(3) and C(4) Cycle Operation

Ecotypic differences in the photosynthetic carbon metabolism of Mollugo verticillata were studied. Variations in C₃ and C₄ cycle activity are apparently due to differences in the activities of enzymes associated with each pathway. Compared to C₄ plants, the activities of C₄ pathway enzymes were generally lower in M. verticillata, with the exception of the decarboxylase enzyme, NAD malic enzyme. The combined total carboxylase enzyme activity of M. verticillata was greater than that of C₃ plants, possibly accounting for the high photosynthetic rates of this species. Unlike either C₃ or C₄ plants, ribulose bisphosphate carboxylase was present in both mesophyll and bundle sheath cell chloroplasts in M. verticillata. The localization of this enzyme in both cells in this plant, in conjunction with an efficient C₄ acid decarboxylation mechanism most likely localized in bundle sheath cell mitochondria, may account for intermediate photorespiration levels previously observed in this species.     

Temperature Effects on Phosphoenolpyruvate Carboxylase from a CAM and a C(4) Plant : A Comparative Study

The effect of temperature in the range from 10 to 35°C on various characteristics of phosphoenolpyruvate carboxylase from the leaves of a CAM plant, Crassula argentea and a C₄ plant Zea mays shows a number of different effects related to the environment in which these distinct types of metabolic specialization normally operate. The Arrhenius plot of V_max for the two enzyme forms shows that the CAM enzyme has a linear increase with temperature while the C₄ enzyme has an inflection at 27°C implying a conformational or aggregational change in the enzyme or a shift in reaction mechanism to one requiring a lower activation energy. The Arrhenius plot of K_m for the two enzymes reveals the startling fact that at temperatures above 20°C an increasing temperature causes an increase in K_mPEP for the CAM enzyme while the C₄ enzyme displays a decreased K_m as the temperature increases. The inhibitory effect of 5 millimolar malate also shows opposite trends for the two enzymes. For the CAM enzyme the percent inhibition by malate increases from essentially none at 15°C to 70% at 35°C. For the C₄ enzyme the percent inhibition drops from about 60% at 20°C to 2% at 30°C. Similar opposite behavior of the two enzymes is found with the K_i for malate. Pretreatment at high temperatures for periods up to 2 hours was found to result in differences similar to those described above if the treated enzyme were subsequently assayed at 25°C.     

Xanthine oxidoreductase is a critical mediator of cigarette smoke-induced endothelial cell DNA damage and apoptosis

Cigarette smoke (CS) exposure is unquestionably the most frequent cause of emphysema in the United States. Accelerated pulmonary endothelial cell (EC) apoptosis is an early determinant of lung destruction in emphysema. One of the pathogenic causes of emphysema is an alveolar oxidant and antioxidant imbalance. The enzyme xanthine oxidoreductase (XOR) has been shown to be a source of reactive oxygen species (ROS) in a multitude of diseases (S. Sakao et al., FASEB J. 21, 3640–3652; 2007). The contribution of XOR to CS-induced apoptosis is not well defined. Here we demonstrate that C57/bl6 mice exposed to CS have increased pulmonary XOR activity and protein levels compared to filtered-air-exposed controls. In addition, we demonstrate that primary pulmonary human lung microvascular endothelial cells exposed to cigarette smoke extract undergo increased rates of caspase-dependent apoptosis that are reliant on XOR activity, ROS production, and p53 function/expression. We also demonstrate that exogenous XOR is sufficient to increase p53 expression and induce apoptosis, suggesting that XOR is an upstream mediator of p53 in CS-induced EC apoptosis. Furthermore, we show that XOR activation results in DNA double-strand breaks that activate the enzyme ataxia telangiectasia mutated, which phosphorylates histone H2AX and upregulates p53. In conclusion, CS increases XOR expression, and the enzyme is both sufficient and necessary for p53 induction and CS-induced EC apoptosis.     

Evolution of C4 Photosynthesis in Flaveria Species : Isoforms of NADP-Malic Enzyme

NADP-malic enzyme (NADP-ME, EC 1.1.1.40), a key enzyme in C₄ photosynthesis, provides CO₂ to the bundle-sheath chloroplasts, where it is fixed by ribulose-1,5-bisphosphate carboxylase/oxygenase. We characterized the isoform pattern of NADP-ME in different photosynthetic species of Flaveria (C₃, C₃-C₄ intermediate, C₄-like, C₄) based on sucrose density gradient centrifugation and isoelectric focusing of the native protein, western-blot analysis of the denatured protein, and in situ immunolocalization with antibody against the 62-kD C₄ isoform of maize. A 72-kD isoform, present to varying degrees in all species examined, is predominant in leaves of C₃ Flaveria spp. and is also present in stem and root tissue. By immunolabeling, NADP-ME was found to be mostly localized in the upper palisade mesophyll chloroplasts of C₃ photosynthetic tissue. Two other isoforms of the enzyme, with molecular masses of 62 and 64 kD, occur in leaves of certain intermediates having C₄ cycle activity. The 62-kD isoform, which is the predominant highly active form in the C₄ species, is localized in bundle-sheath chloroplasts. Among Flaveria spp. there is a 72-kD constitutive form, a 64-kD form that may have appeared during evolution of C₄ metabolism, and a 62-kD form that is necessary for the complete functioning of C₄ photosynthesis.     

Dark/Light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories

Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO₃⁻ and Mg²⁺ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C₃); P. maximum (C₄ phosphoenolpyruvate carboxykinase); P. milioides (C₃/C₄); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C₃); P. miliaceum (C₄ NAD malic enzyme); Zea mays and Sorghum bicolor (C₄ NADP malic enzyme); Moricandia arvensis (C₃/C₄); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C₃ species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO₂ and Mg²⁺ activation, but which can be converted to an activatable state upon exposure of the leaf to light.