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PubMed2006

Bacterial oxygenases: in vivo enzyme biosensors for organic pollutants.

An in vivo enzyme-based biosensor platform was developed that uses specific oxygenase enzymes to detect aromatic compounds in water. Bacteria capable of degrading highly reduced hydrocarbons initiate substrate oxidation using well-characterised oxygenase enzymes, which due to their specificity, stability and high activity can be applied in vivo as biosensor components. Oxygenase enzyme activity was determined in vivo using BD Oxygen Biosensor plates to measure oxygenase-mediated oxygen depletion in the presence of specific aromatic analytes. The BTEX family of compounds (benzene, toluene, ethylbenzene and the three isomers of xylene) were used as model compounds. Detection limits and sensitivity achieved using this approach (microM detection range) was similar to levels achieved with oxygen electrode-based and some recombinant DNA-based approaches. No significant signals were detected with hydrocarbons that were not substrates of the initial oxygenases.

Bacteria, Aerobic↗
PubMed2006

Genetic analysis of the balhimycin (vancomycin-type) oxygenase genes.

In the balhimycin biosynthesis three oxygenases OxyA, OxyB and OxyC are responsible for the oxidative phenol coupling reactions, which lead to the ring-closures between the aromatic amino acid side chains in the heptapeptide aglycone. These ring-closures constrain the peptide backbone into the cup-shaped conformation that is required for binding to the Lys-D-Ala-D-Ala-terminus of the cell wall precursor peptide and represent one of the essential features of glycopeptide antibiotics. In the balhimycin biosynthetic gene cluster the oxygenase genes oxyA, oxyB and oxyC have been identified downstream of the peptide synthetase genes. Reverse transcription (RT)-PCR analyses revealed that these oxygenase genes in Amycolatopsis balhimycina are co-transcribed. Non-polar mutants (NPoxyA, DeltaoxyB and DeltaoxyC) were constructed, cultivated in production medium and assayed for the presence of glycopeptides and glycopeptide precursors by HPLC-ESI-MS. The mutant NPoxyA produces mainly monocyclic, the mutant DeltaoxyB linear and the mutant DeltaoxyC bicyclic peptides. These results definitely confirm the sequence of the three oxidative ring-closing steps (OxyB-OxyA-OxyC). The heterologous complementation of the mutant strains with the corresponding oxygenase genes from the vancomycin producer A. orientalis restored the production of balhimycin, which proves the functional equivalence of the oxygenases from the balhimycin and vancomycin producer. For the first time it is now possible to combine the genetic data obtained from the balhimycin producer with the biochemical and structural data obtained from the vancomycin producer.

Actinomycetales↗
PubMed2005

Heme oxygenase attenuates oxidative stress and inflammation, and increases VEGF expression in portal hypertensive rats.

BACKGROUND/AIMS: The pathophysiological significance of heme oxygenase-1 up-regulation in portal hypertension is not completely understood. In this study, we determined the role of heme oxygenase-1 on oxidative stress, inflammation, angiogenesis, and splanchnic hemodynamics in rats with portal hypertension induced by partial portal vein ligation. METHODS: Rats were treated with the heme oxygenase inhibitor SnMP or vehicle for 7 days. Then, oxidative stress was quantified by superoxide anion production, and inflammatory response was assessed by immunofluorescence. Expression of angiogenesis mediators was determined by western blotting, and the extent of portosystemic collaterals by radioactive microspheres. Hemodynamic studies were performed by flowmetry. RESULTS: Oxidative stress was significantly increased in the mesentery of portal hypertensive rats, as compared with sham-operated controls. In portal hypertensive rats, chronic heme oxygenase inhibition (1) potentiated oxidative stress and inflammation, (2) significantly decreased VEGF expression, without modifying the extent of collaterals or the splanchnic neovascularization, and (3) significantly decreased superior mesenteric artery blood flow and portal pressure. CONCLUSIONS: This study demonstrates that heme oxygenase plays an important (beneficial) role attenuating oxidative stress and inflammation, but it also plays a detrimental role in stimulating VEGF production, and contributing to the development of hyperdynamic splanchnic circulation in rats with portal hypertension.

Animals↗
PubMed2005

Structure of the terminal oxygenase component of angular dioxygenase, carbazole 1,9a-dioxygenase.

Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. This reaction is an initial degradation reaction of the carbazole degradation pathway by various bacterial strains. Only a limited number of Rieske non-heme iron oxygenase systems (ROSs) can catalyze this novel reaction, termed angular dioxygenation. Angular dioxygenation is also involved in the degradation pathways of carbazole-related compounds, dioxin, and CARDO can catalyze the angular dioxygenation for dioxin. CARDO consists of a terminal oxygenase component (CARDO-O), and the electron transport components, ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R). CARDO-O has a homotrimeric structure, and governs the substrate specificity of CARDO. Here, we have determined the crystal structure of CARDO-O of Janthinobacterium sp. strain J3 at a resolution of 1.95A. The alpha3 trimeric overall structure of the CARDO-O molecule roughly corresponds to the alpha3 partial structures of other terminal oxygenase components of ROSs that have the alpha3beta3 configuration. The CARDO-O structure is a first example of the terminal oxygenase components of ROSs that have the alpha3 configuration, and revealed the presence of the specific loops that interact with a neighboring subunit, which is proposed to be indispensable for stable alpha3 interactions without structural beta subunits. The shape of the substrate-binding pocket of CARDO-O is markedly different from those of other oxygenase components involved in naphthalene and biphenyl degradation pathways. Docking simulations suggested that carbazole binds to the substrate-binding pocket in a manner suitable for catalysis of angular dioxygenation.

Bacterial Proteins↗
PubMed2002

Spinal cord heme oxygenase participates in glutamate-induced pain-related behaviors.

Heme oxygenase catalyzes the formation of CO, Fe(2+) and biliverdin from the substrate heme. In these studies, we attempted to define the roles heme oxygenase play in pain-related behaviors induced by intrathecal injection of the spinal neurotransmitter glutamate. The intrathecal injection of glutamate or the more selective agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in C57Bl/6 mice lead to caudally directed pain behaviors which were sensitive to the heme oxygenase inhibitors tin protoporphyrin (Sn-protoporphyrin) and chromium mesoporphyrin (Cr-mesoporphyrin). Intrathecal injections of glutamate in heme oxygenase type 2 (HO-2) null-mutant animals resulted in reduced pain-related behaviors when compared with wild type animals. Glutamate, NMDA and AMPA stimulated cGMP accumulation in mouse spinal cord slices, which was blocked by heme oxygenase inhibitors. Glutamate did not stimulate cGMP production in HO-2 null-mutant animals. Our data are consistent with the hypothesis that pain-related behaviors induced by spinal glutamate rely on the activation of HO-2 and subsequent production of cGMP.

Animals↗
PubMed2000

Alteration of expression levels of neuronal nitric oxide synthase and haem oxygenase-2 messenger RNA in the hippocampi and cortices of young adult and aged cognitively unimpaired and impaired Long-Evans rats.

Neuronal nitric oxide synthase and haem oxygenase-2 are postulated to be important enzymes involved in neuronal transmission and modulation of free radical levels in neurons. Hippocampal and cortical neuronal nitric oxide synthase and haem oxygenase-2 expressions were compared in young adult (6 months) and aged (24-26 months) Long-Evans rats. Aged rats were assigned as either cognitively unimpaired or impaired based on their performances in the Morris water maze behavioural task. In situ hybridization revealed increased neuronal nitric oxide synthase messenger RNA levels in selected regions of the hippocampi and cortices of aged rats. Moreover, aged cognitively impaired animals showed significantly higher neuronal nitric oxide synthase messenger RNA expression than aged cognitively unimpaired animals in several brain regions. For haem oxygenase-2 mRNA expressions, both young and aged cognitively impaired rats showed increased expressions in hippocampi compared with aged cognitively unimpaired rats, while no difference was found in cortices between all three animal groups. The increase in neuronal nitric oxide synthase messenger RNA expression levels in the aged animals may be related to increased free radical production occurring in ageing. Alternatively, elevated neuronal nitric oxide synthase and haem oxygenase-2 messenger RNA expressions may represent compensatory responses to oxidative stress and age-related changes in neuronal functions. Regarding cognitive status, aged cognitively impaired rats showed significant spatial memory deficits relative to young and aged cognitively unimpaired rats. Our data suggest a correlation between age-related cognitive impairment and change in messenger RNA expressions for the neuronal nitric oxide synthase and haem oxygenase-2 systems in brain areas implicated in learning and memory processes.

Aging↗
PubMed2003

Purification and gene cloning of the oxygenase component of the terephthalate 1,2-dioxygenase system from Delftia tsuruhatensis strain T7.

The terephthalate 1,2-dioxygenase system (TERDOS) was found in cell extracts of Delftia tsuruhatensis strain T7 (=IFO16741) grown in terephthalate-salt medium. The cell extract was separated by anion exchange chromatography to yield two fractions (R and Z) that were necessary for oxygenation of terephthalate with NADH and Fe(2+). The oxygenase component of TERDOS (TerZ) was purified from fraction Z by gel filtration chromatography to near homogeneity. An alpha(3)beta(3) subunit structure was deduced from the molecular masses of 235, 46 and 17 kDa of the native complex and the alpha- and beta-subunits, respectively. The N-terminal amino acid sequences of the two subunits of TerZ allowed polymerase chain reaction primers to be deduced and the DNA sequence of the alpha-subunit was determined. The amino acid sequence of the alpha-subunit (TerZalpha) showed significant similarities to the large subunits of multicomponent ring-hydroxylating oxygenases. Two motifs in the deduced amino acid sequence, a Rieske [2Fe-2S] center and a mononuclear Fe(II) binding site, were observed. Phylogenetic analyses indicated that TerZalpha and the large oxygenase component subunits ortho-halobenzoate 1,2-dioxygenase and salicylate-5-hydroxylase form a cluster that is distant from the rest of the large oxygenase subunits of multicomponent ring-hydroxylating oxygenases.

Amino Acid Sequence↗
PubMed1995

Reconstitution of the second step in NO synthesis using the isolated oxygenase and reductase domains of macrophage NO synthase.

Inducible macrophage NO synthase (iNOS) is a homodimer of 130 kDa subunits. Trypsinolysis of iNOS inactivates its NO synthesis activity and cleaves the enzyme into a dimeric oxygenase fragment that contains heme, tetrahydrobiopterin, and the substrate binding site and a monomeric reductase fragment that contains FAD, FMN, calmodulin, and the binding site for NADPH [Ghosh, D. I., & Stuehr, D. H. (1995) Biochemistry 34, 801-807]. In this paper, we describe the reconstitution of NO synthesis activity utilizing the isolated oxygenase and reductase domains of iNOS. Mixing the domains at various ratios showed that NO was not produced from L-arginine but could be formed from the reaction intermediate N omega-hydroxy-L-arginine (L-NOHA). The apparent Km with L-NOHA in the reconstituted system was 100 microM versus 19 microM for native iNOS. D-NOHA was not a substrate. Maximum specific activity (per heme) occurred at an oxygenase to reductase molar ratio of 4:1, with higher ratios causing some inhibition. Reconstitution of activity was associated with electron transfer between the domain fragments and led to an incomplete reduction of the oxygenase domain heme iron. L-NOHA, but not L-arginine, increased NADPH consumption in the reconstituted system. Between 2.5 and 3.0 NADPH were consumed per NO formed from L-NOHA, considerably higher than the stoichiometry obtained with native iNOS (0.5 NADPH oxidized per NO formed), indicating an uncoupled electron transfer between the domain fragments. Thus, the isolated iNOS reductase and oxygenase domains each retain their separate catalytic functions but interact to catalyze only the second step of NO synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine↗
PubMed1993

The oxygenase reaction of acetolactate synthase.

In addition to the physiological reactions catalyzed by acetolactate synthase, it supports an oxygen-consuming side reaction. Although the synthase and oxygenase activities are activated to somewhat different extents by various metals (Mn2+, Mg2+, Ca2+, Co2+, Zn2+, Ni2+, Cd2+, Cu2+, Ba2+, Al3+), the modest degree of these differences (at most 6-fold) and the high degree of promiscuity of the enzyme with respect to its metal requirement suggest that the metal is not intimately involved in the chemistry of either reaction. Saturation of the oxygenase reaction occurs at pyruvate concentrations below the limit of sensitivity for the oxygen electrode (< 10 microM), at higher concentrations pyruvate inhibits the rate of oxygen consumption. At a noninhibitory concentration of pyruvate (1 mM), inhibition of the reaction is also observed with alpha-ketobutyrate. Inhibition of the oxygenase reaction by high concentrations of pyruvate or alpha-ketobutyrate is presumably due to competition between these substrates and molecular oxygen for a common carbanionic reaction intermediate, the conjugate base of (hydroxyethyl)thiamin pyrophosphate. Inhibition of the reaction indicates that the lactylthiamin pyrophosphate intermediate can decarboxylate prior to binding of the second pyruvate or alpha-ketobutyrate. At high concentrations of pyruvate or alpha-ketobutyrate, only incomplete inhibition of the oxygenase reaction is achieved (65-89% or 89-93% maximal inhibition, respectively). This incomplete inhibition of the oxygenase reaction by alpha-keto acids indicates that the reaction is not Theorell-Chance with respect to addition of the second alpha-keto acid and that oxygen has more than one route of access to the carbanionic reaction intermediate.

Acetolactate Synthase↗
PubMed2006

Fungal heme oxygenases: Functional expression and characterization of Hmx1 from Saccharomyces cerevisiae and CaHmx1 from Candida albicans.

Heme oxygenases convert heme to free iron, CO, and biliverdin. Saccharomyces cerevisiae and Candida albicans express putative heme oxygenases that are required for the acquisition of iron from heme, a critical process for fungal survival and virulence. The putative heme oxygenases Hmx1 and CaHmx1 from S. cerevisiae and C. albicans, respectively, minus the sequences coding for C-terminal membrane-binding domains, have been expressed in Escherichia coli. The C-terminal His-tagged, truncated enzymes are obtained as soluble, active proteins. Purified ferric Hmx1 and CaHmx1 have Soret absorption maxima at 404 and 410 nm, respectively. The apparent heme binding Kd values for Hmx1 and CaHmx1 are 0.34 +/- 0.09 microM and 1.0 +/- 0.2 microM, respectively. The resonance Raman spectra of Hmx1 reveal a heme binding pocket similar to those of the mammalian and bacterial heme oxygenases. Several reductants, including ascorbate, yeast cytochrome P450 reductase (CPR), human CPR, spinach ferredoxin/ferredoxin reductase, and putidaredoxin/putidaredoxin reductase, are able to provide electrons for biliverdin production by Hmx1 and CaHmx1. Of these, ascorbate is the most effective reducing partner. Heme oxidation by Hmx1 and CaHmx1 regiospecifically produces biliverdin IXalpha. Spectroscopic analysis of aerobic reactions with H2O2 identifies verdoheme as a reaction intermediate. Hmx1 and CaHmx1 are the first fungal heme oxygenases to be heterologously overexpressed and characterized. Their heme degradation activity is consistent with a role in iron acquisition.

Amino Acid Sequence↗
PubMed2002

Oxidation of heme to beta- and delta-biliverdin by Pseudomonas aeruginosa heme oxygenase as a consequence of an unusual seating of the heme.

The origin of the unusual regioselectivity of heme oxygenation, i.e. the oxidation of heme to delta-biliverdin (70%) and beta-biliverdin (30%), that is exhibited by heme oxygenase from Pseudomonas aeruginosa (pa-HO) has been studied by (1)H NMR, (13)C NMR, and resonance Raman spectroscopies. Whereas resonance Raman indicates that the heme-iron ligation in pa-HO is homologous to that observed in previously studied alpha-hydroxylating heme oxygenases, the NMR spectroscopic studies suggest that the heme in this enzyme is seated in a manner that is distinct from that observed for all other alpha-hydroxylating heme oxygenase enzymes for which a structure is known. In pa-HO, the heme is rotated in-plane approximately 110 degrees, so the delta-meso-carbon of the major orientational isomer is located within the HO-fold in the place where the alpha-hydroxylating enzymes typically place the alpha-meso-carbon. The unusual heme seating displayed by pa-HO places the heme propionates so that these groups point in the direction of the solvent-exposed heme edge and appears to originate in large part from the absence of stabilizing interactions between the polypeptide and the heme propionates, which are typically found in alpha-hydroxylating heme oxygenase enzymes. These interactions typically involve Lys-16 and Tyr-112, in Neisseriae meningitidis HO, and Lys-16 and Tyr-134, in human and rat HO-1. The corresponding residues in pa-HO are Asn-19 and Phe-117, respectively. In agreement with this hypothesis, we found that the Asn-19 Lys/Phe-117 Tyr double mutant of pa-HO exists as a mixture of molecules exhibiting two distinct heme seatings; one seating is identical to that exhibited by wild-type pa-HO, whereas the alternative seating is very similar to that typical of alpha-hydroxylating heme oxygenase enzymes and is related to the wild-type seating by approximately 110 degrees in-plane rotation of the heme. Furthermore, each of these heme seatings in the pa-HO double mutant gives rise to a subset of two heme isomeric orientations that are related to each other by 180 degrees rotation about the alpha-gamma-meso-axis. The coexistence of these molecules in solution, in the proportions suggested by the corresponding area under the peaks in the (1)H NMR spectrum, explains the unusual regioselectivity of heme oxygenation observed with the double mutant, which we found produces alpha- (55%), delta- (35%), and beta-biliverdin (10%). Alpha-biliverdin is obtained by oxidation of the heme seated similar to that of alpha-hydroxylating enzymes, whereas beta- and delta-biliverdin are formed from the oxidation of heme seated as in wild-type pa-HO.

Biliverdine↗
PubMed2000

Phytobilin biosynthesis: the Synechocystis sp. PCC 6803 heme oxygenase-encoding ho1 gene complements a phytochrome-deficient Arabidopsis thalianna hy1 mutant.

The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IXalpha, in a reaction catalyzed by heme oxygenase. An Arabidopsis thaliana hy1 mutant was previously shown to be deficient in phytochrome responses, and these responses were regained when the plants were administered biliverdin IXalpha. A heme oxygenase-encoding gene, ho1, was recently cloned from the cyanobacterium Synechocystis sp. PCC 6803. When ho1 was expressed in Escherichia coli, the cells produced active ferredoxin-dependent soluble heme oxygenase. The open reading frame of ho1 was fused in frame with a chloroplast transit peptide-encoding sequence from the oli gene of Antirrhinum majus. This construct was placed in a binary plasmid vectorcontaining a kanamycin resistance marker and a cauliflower mosaic virus 35S promoter to control expression of the chimeric oli-ho1 gene and used to transform A. thaliana hy1 plants. Two independent transformed lines were obtained that had the phenotype of the parental Landsberg erecta line and expressed the chimeric gene, as indicated by detection of its mRNA by reverse transcriptase-polymerase chain reaction. The results indicate that Synechocystis sp. PCC 6803 heme oxygenase encoded by ho1 can substitute for the defective HY1 gene product and that the only required enzyme activity of the HY1 gene product is heme oxygenase.

Amino Acid Sequence↗
PubMed1976

Induction of benzo[a]pyrene Mono-oxygenase in liver cell culture by the photochemical generation of active oxygen species. Evidence for the involvement of singlet oxygen and the formation of a stable inducing intermediate.

1. The photochemical generation of excited states of oxygen in liver cell culture by the mild ilumination of culture medium containing riboflavin, results in stimulation of benzo[a]pyrene 3-mono-oxygenase, a cytochrome P-450-linked mono-oxygenase. 2. The same large increase in mono-oxygenase activity was found when medium containing riboflavin was illuminated in the absence of cells and then stored in the dark for 24h before contact with the cells. From this it may be inferred that stimulation is due to the formation of a stable inducer in the culture medium. Further experiments indicate that the stable inducer is due to the photo-oxidation of an amino acid. 3. Evidence that singlet oxygen is responsible for initiating the stimulation of the mono-oxygenase is based on the use of molecules that scavenge particular active oxygen species. Of all the scavengers tested, only those that scavenge single oxygen inhibited the stimulation. 4. A hypothesis is developed to relate the stimulation of the mono-oxygenase by singlet oxygen in cultured cells to the regulation of the cytochrome P-450 enzyme system in vivo. It is suggested that single oxygen generation within cells may be a common factor linking the many structurally diverse inducers of the enzyme system.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed1983

The influence of some methodological factors on measurement of tryptophan oxygenase activities in crude homogenates of rat liver.

1. With two different methods for assaying the tryptophan oxygenase activity in rat liver homogenates, the effects of some methodological factors on the activity of the enzyme were studied. 2. In fed, but not in starved, rats a compound(s) absorbing at 365 nm, interfering with the reading of kynurenine absorbance, disappeared gradually during incubation. 3. A correction for this tryptophan-independent reaction was necessary in order to determine correct tryptophan oxygenase activity. 4. Blood remaining in liver tissue post mortem can serve as a source of cofactor haem for tryptophan oxygenase, causing spuriously high values for the activity of the holoenzyme form of tryptophan oxygenase. 5. A rapid and progressive activation of tryptophan oxygenase post mortem occurs in undisrupted liver tissue, and this activation is temperature-dependent.

Animals↗
PubMed1991

A heat-inducible nuclear factor that binds to the heat-shock element of the human haem oxygenase gene.

Haem oxygenase is a heat-shock protein in several rat tissues, as well as in certain human cells such as Hep3B hepatoma cells. In common with other heat-shock-protein genes, both the human and the rat haem oxygenase genes contain a heat-shock element (HSE) in their promoter regions. In the present study we have identified a factor in nuclear extracts of human Hep3B cells which binds specifically to the HSE of the human haem oxygenase gene. The factor in Hep3B cells was significantly induced within 1 h after heat-shock treatment, and the induction was blocked by treatment of cells with actinomycin D or cycloheximide. The factor was not detected in human HepG2 hepatoma cells, which exhibit the heat-mediated induction of heat-shock protein 70 mRNA, but not that of haem oxygenase mRNA. These findings suggest that the heat-inducible nuclear factor is increased at the level of transcription and that it may activate the human haem oxygenase gene via the HSE after heat treatment.

Base Sequence↗
PubMed2004

Generation of bile pigments by haem oxygenase: a refined cellular strategy in response to stressful insults.

The family of haem oxygenase enzymes is unique in nature for its role in haem degradation. Haem is cleaved at the alpha-meso position by haem oxygenase with the support of electrons donated by cytochrome P450 reductase, the first products of this reaction being CO, iron and biliverdin. Biliverdin is then converted to bilirubin by biliverdin reductase. If haem is viewed as a substrate for an anabolic pathway, it becomes evident that haem oxygenases do not break down haem for elimination from the body, but rather use haem to generate crucial molecules that can modulate cellular functions. The facts that biliverdin and bilirubin are potent antioxidants and that CO is both a vasoactive and signalling molecule sustain this idea. The existence of a constitutive haem oxygenase (HO-2), mainly present in the vasculature and nervous system, and an inducible haem oxygenase (HO-1), which is highly expressed during stress conditions in all tissues, also suggests that cells have evolved a fine control of this enzymic pathway to ultimately regulate haem consumption and to ensure production of CO, biliverdin/bilirubin and iron during physiological and pathophysiological situations. This review will focus primarily on the biological actions of biliverdin and bilirubin derived from the haem oxygenase/biliverdin reductase systems and their potential roles in counteracting oxidative and nitrosative stress.

Animals↗
PubMed1998

Phytobilin biosynthesis: cloning and expression of a gene encoding soluble ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803.

The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IX alpha, in a reaction catalyzed by heme oxygenase. A gene containing an open reading frame with a predicted polypeptide that has a sequence similar to that of a conserved region of animal microsomal heme oxygenases was identified in the published genomic sequence of Synechocystis sp. PCC 6803. This gene, named ho1, was cloned and expressed in Escherichia coli under the control of the lacZ promoter. Cells expressing the gene became green colored due to the accumulation of biliverdin IX alpha. The size of the expressed protein was equal to the predicted size of the Synechocystis gene product, named HO1. Heme oxygenase activity was assayed in incubations containing extract of transformed E. coli cells. Incubations containing extract of induced cells, but not those containing extract of uninduced cells, had ferredoxin-dependent heme oxygenase activity. With mesoheme as the substrate, the reaction product was identified as mesobiliverdin IX alpha by spectrophotometry and reverse-phase HPLC. Heme oxygenase activity was not sedimented by centrifugation at 100, 000 g. Expression of HO1 increased several-fold during incubation of the cells for 72 h in iron-deficient medium.

Bacterial Proteins↗
PubMed1994

Heme oxygenase 1 mediates an adaptive response to oxidative stress in human skin fibroblasts.

Oxidative stress of human skin fibroblasts by treatment with ultraviolet A (UVA) radiation has been shown to lead to an increase in levels of the heme catabolizing enzyme heme oxygenase 1 [heme, hydrogen-donor:oxygen oxidoreductase (alpha-methene-oxidizing, hydroxylating), EC 1.14.99.3] and the iron storage protein ferritin. Here we show that human skin fibroblasts, preirradiated with UVA, sustain less membrane damage during a subsequent exposure to UVA radiation than cells that had not been preirradiated. Pretreating cells with heme oxygenase 1 antisense oligonucleotide inhibited the irradiation-dependent induction of both the heme oxygenase I enzyme and ferritin and abolished the protective effect of preirradiation. Inhibition of the UVA preirradiation-dependent increase in ferritin, but not heme oxygenase, with desferrioxamine also abolished the protection. This identifies heme oxygenase 1 as a crucial enzymatic intermediate in an oxidant stress-inducible antioxidant defense mechanism, involving ferritin, in human skin fibroblasts.

Adaptation, Physiological↗