Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide.
(1/94)
The flavohaemoglobin Hmp of Escherichia coli is inducible by nitric oxide (NO) and provides protection both aerobically and anaerobically from inhibition of growth by NO and agents that cause nitrosative stress. Here we report rapid kinetic studies of NO binding to Fe(III) Hmp with a second order rate constant of 7.5 x 10(5) M(-1) s(-1) to generate a nitrosyl adduct that was stable anoxically but decayed in the presence of air to reform the Fe(III) protein. NO displaced CO bound to dithionite-reduced Hmp but, remarkably, CO recombined after only 2 s at room temperature indicative of NO reduction and dissociation from the haem. Addition of NO to anoxic NADH-reduced Hmp also generated a nitrosyl species which persisted while NADH was oxidised. These results are consistent with direct demonstration by membrane-inlet mass spectrometry of NO consumption and nitrous oxide production during anoxic incubation of NADH-reduced Hmp. The results demonstrate a new mechanism by which Hmp may eliminate NO under anoxic growth conditions. (+info)
Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence.
(2/94)
Three agr-like genes (fsrA, fsrB, and fsrC, for Enterococcus faecalis regulator) were found upstream of the previously reported gelatinase gene (gelE) and a downstream putative serine protease gene (sprE; accession number Z12296) of Enterococcus faecalis OG1RF. The deduced amino acid sequence of fsrA shows 26% identity and 38% similarity to Staphylococcus aureus AgrA (the response regulator of the accessory gene regulator system in the agr locus), FsrB shows 23% identity and 41% similarity to S. aureus AgrB, and FsrC shows 23% identity and 36% similarity to S. aureus AgrC (the sensor transducer of Agr system). Northern blot analysis suggested that gelE and sprE are cotranscribed and that fsrB and fsrC are also cotranscribed in OG1RF. Northern blot analysis of fsrA, fsrB, fsrC, gelE, and sprE insertion mutants showed that fsrB, fsrC, gelE, and sprE are not expressed in fsrA, fsrB, and fsrC mutants, while insertion in an open reading frame further upstream of fsrA did not effect the expression of these genes, suggesting that agr-like genes may be autoregulated and that they regulate gelE and sprE expression, as further confirmed by complementation of fsr gene mutations with a 6-kb fragment which contains all three fsr genes in the shuttle vector, pAT18. Testing of 95 other isolates of E. faecalis showed that 62% produced gelatinase (Gel(+)), while 91% (including all Gel(+) strains) hybridized to a gelE probe; 71% (including all Gel(+) strains) hybridized to an fsr probe, corroborating the conclusion that both gelE and fsr are necessary for gelatinase production. Testing of fsrA, fsrB, and sprE mutants in a mouse peritonitis model showed that sprE and agr-like gene mutants resulted in highly significantly prolonged survival compared to the parent strain OG1RF, a finding similar to what we had previously shown for a gelE mutant. These results suggest that sprE and agr-like genes contribute to the virulence of E. faecalis OG1RF in this model. (+info)
Contrasting effects of N5-substituted tetrahydrobiopterin derivatives on phenylalanine hydroxylase, dihydropteridine reductase and nitric oxide synthase.
(3/94)
Tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H(4)biopterin] is one of several cofactors of nitric oxide synthases (EC 1.14.13.39). Here we compared the action of N(5)-substituted derivatives on recombinant rat neuronal nitric oxide synthase with their effects on dihydropteridine reductase (EC 1.6.99.7) and phenylalanine hydroxylase (EC 1.14.16.1),the well-studied classical H(4)biopterin-dependent reactions. H(4)biopterin substituted at N(5) with methyl, hydroxymethyl, formyl and acetyl groups were used. Substitution at N(5) occurs at a position critical to the redox cycle of the cofactor in phenylalanine hydroxylase/dihydropteridine reductase. We also included N(2)'-methyl H(4)biopterin, a derivative substituted at a position not directly involved in redox cycling, as a control. As compared with N(5)-methyl H(4)biopterin, N(5)-formyl H(4)biopterin bound with twice the capacity but stimulated nitric oxide synthase to a lesser extent. Depending on the substituent used, N(5)-substituted derivatives were redox-active: N(5)-methyl- and N(5)-hydroxyl methyl H(4)biopterin, but not N(5)-formyl- and N(5)-acetyl H(4)biopterin, reduced 2,6-dichlorophenol indophenol. N(5)-Substituted H(4)biopterin derivatives were not oxidized to products serving as substrates for dihydropteridine reductase and,depending on the substituent, were competitive inhibitors of phenylalanine hydroxylase: N(5)-methyl- and N(5)-hydroxymethyl H(4)biopterin inhibited phenylalanine hydroxylase, whereas N(5)-formyl- and N(5)-acetyl H(4)biopterin had no effect. Our data demonstrate differences in the mechanism of stimulation of phenylalanine hydroxylase and nitric oxide synthase by H(4)biopterin. They are compatible with a novel, non-classical, redox-active contribution of H(4)biopterin to the catalysis of the nitric oxide synthase reaction. (+info)
Flavohemoglobin Hmp affords inducible protection for Escherichia coli respiration, catalyzed by cytochromes bo' or bd, from nitric oxide.
(4/94)
Respiration of Escherichia coli catalyzed either by cytochrome bo' or bd is sensitive to micromolar extracellular NO; extensive, transient inhibition of respiration increases as dissolved oxygen tension in the medium decreases. At low oxygen concentrations (25-33 microm), the duration of inhibition of respiration by 9 microm NO is increased by mutation of either oxidase. Respiration of an hmp mutant defective in flavohemoglobin (Hmp) synthesis is extremely NO-sensitive (I(50) about 0.8 microm); conversely, cells pre-grown with sodium nitroprusside or overexpressing plasmid-borne hmp(+) are insensitive to 60 microm NO and have elevated levels of immunologically detectable Hmp. Purified Hmp consumes O(2) at a rate that is instantaneously and extensively (>10-fold) stimulated by NO due to NO oxygenase activity but, in the absence of NO, Hmp does not contribute measurably to cell oxygen consumption. Cyanide binds to Hmp (K(d) 3 microm). Concentrations of KCN (100 microm) that do not significantly inhibit cell respiration markedly suppress the protection of respiration from NO afforded by Hmp and abolish NO oxygenase activity of purified Hmp. The results demonstrate the role of Hmp in protecting respiration from NO stress and are discussed in relation to the energy metabolism of E. coli in natural O(2)-depleted environments. (+info)
Molecular characterization of Drosophila melanogaster dihydropteridine reductase.
(5/94)
Dihydropteridine reductase (DHPR) catalyzes the NAD(P)H-mediated reduction of quinonoid dihydropteridine as a part of pterin-dependent aromatic amino acid hydroxylation. We isolated a fragment of Drosophila DHPR gene by PCR using degenerate primers. By screening a cDNA library, we obtained full-length clones. The predicted amino acid sequence of the Drosophila DHPR protein was highly homologous to other species including human and mouse. In particular, the Tyr-(Xaa)(3)-Lys motif, known as the NAD(P)H binding domain, and most amino acids relevant to quinonoid dihydropteridine binding site are identical to human DHPR. The recombinant DHPR protein expressed in Escherichia coli showed DHPR enzyme activity. Northern blot analysis revealed two transcripts of 1.1 and 0.9 kb. Genomic DNA sequencing revealed that the gene consists of two exons interrupted by a single 96-bp intron. The two transcripts have alternative promoters, both having no putative TATA box or CAAT box, but sharing a common poly(A)(+) signal. The existence of two alternative promoters suggests that each transcript be regulated independently through different stimuli. Further study is needed to examine the expression and function of the two alternative transcripts. (+info)
Flavohemoglobin, a globin with a peroxidase-like catalytic site.
(6/94)
Biochemical studies of flavohemoglobin (Hmp) from Escherichia coli suggest that instead of aerobic oxygen delivery, a dioxygenase converts NO to NO3(-) and anaerobically, an NO reductase converts NO to N(2)O. To investigate the structural features underlying the chemical reactivity of Hmp, we have measured the resonance Raman spectra of the ligand-free ferric and ferrous protein and the CO derivatives of the ferrous protein. At neutral pH, the ferric protein has a five-coordinate high-spin heme, similar to peroxidases. In the ferrous protein, a strong iron-histidine stretching mode is present at 244 cm(-1). This frequency is much higher than that of any other globin discovered to date, although it is comparable to those of peroxidases, suggesting that the proximal histidine has imidazolate character. In the CO derivative, an open and a closed conformation were detected. The distal environment of the closed conformation is very polar, where the heme-bound CO strongly interacts with the B10 Tyr and/or the E7 Gln. These data demonstrate that the active site structure of Hmp is very similar to that of peroxidases and is tailored to perform oxygen chemistry. (+info)
Escherichia coli flavohaemoglobin (Hmp) with equistoichiometric FAD and haem contents has a low affinity for dioxygen in the absence or presence of nitric oxide.
(7/94)
A purification procedure for flavohaemoglobin Hmp (NO oxygenase) is described that gives high yields of protein with equistoichiometric haem and FAD contents. H(2)O(2) accumulated on NADH oxidation by the purified protein and in cell extracts with elevated Hmp contents. H(2)O(2) probably arose by dismutation from superoxide, which was also detectable during oxygen reduction; water was not a product. In the absence of agents that scavenge superoxide and peroxide, the mean K(m) for oxygen was 80 microM; the addition of 15 microM FAD decreased the K(m) for oxygen to 15 microM without a change in V(max) but catalysed cyanide-insensitive oxygen consumption, attributed to electron transfer from flavins to O(2). Purified Hmp consumed NO in the absence of added FAD (approx. 1 O(2) per NO), which is consistent with NO oxygenation. However, half-maximal rates of NO-stimulated O(2) consumption required approx. 47 microM O(2); NO removal was ineffective at physiologically relevant O(2) concentrations (below approx. 30 microM O(2)). On exhaustion of O(2), NO was removed by a cyanide-sensitive process attributed to NO reduction, with a turnover number approx. 1% of that for oxygenase activity. These results suggest that the ability of Hmp to detoxify NO might be compromised in hypoxic environments. (+info)
Diagnosis of dopa-responsive dystonia and other tetrahydrobiopterin disorders by the study of biopterin metabolism in fibroblasts.
(8/94)
BACKGROUND: Dopa-responsive dystonia (DRD) and tetrahydrobiopterin (BH4) defects are inherited disorders characterized by monoamine neurotransmitter deficiency with decreased activity of one of the BH4-metabolizing enzymes. The aim of the study was to determine the utility of cultured skin fibroblasts for the diagnosis of these diseases. METHODS: Neopterin and biopterin production and GTP cyclohydrolase I (GTPCH) activity were measured in cytokine-stimulated fibroblasts; 6-pyruvoyltetrahydropterin synthase (PTPS), sepiapterin reductase (SR), and dihydropteridine reductase (DHPR) activities were measured in unstimulated fibroblasts. We examined 8 patients with DRD, 3 with autosomal recessive GTPCH deficiency, 7 with PTPS deficiency, 3 with DHPR deficiency, and 49 controls (35 fibroblast and 14 amniocyte samples). RESULTS: Fibroblasts from patients with DRD and autosomal recessive GTPCH deficiency showed reduced GTPCH activity (15.4% and 30.7% of normal activity, respectively) compared with controls (P < 0.001). Neopterin production was very low and biopterin production was reduced in both disorders. PTPS- and DHPR-deficient cells showed no enzyme activities; in PTPS deficiency the pattern of pterin production was typical (neopterin, 334-734 pmol/mg; controls, 18-98 pmol/mg; biopterin, 0 pmol/mg; controls, 154-303 pmol/mg). Reference values of all enzyme activities and pterin production were measured in fibroblasts and also in amniocytes for prenatal diagnosis. CONCLUSIONS: Cultured skin fibroblasts are a useful tool in the diagnosis of BH4 deficiencies. Intracellular neopterin and biopterin concentrations and GTPCH activity in cytokine-stimulated fibroblasts are particularly helpful in diagnosing patients with DRD. (+info)