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Biosynthesis of heme d1, the essential prosthetic group of the dissimilatory nitrite reductase cytochrome cd1, requires the methylation of the tetrapyrrole precursor uroporphyrinogen III at positions C-2 and C-7. We produced Pseudomonas aeruginosa NirE, a putative S-adenosyl-l-methionine (SAM)-dependent uroporphyrinogen III methyltransferase, as a recombinant protein in Escherichia coli and purified it to apparent homogeneity by metal chelate and gel filtration chromatography. Analytical gel filtration of purified NirE indicated that the recombinant protein is a homodimer. NirE was shown to be a SAM-dependent uroporphyrinogen III methyltransferase that catalyzes the conversion of uroporphyrinogen III into precorrin-2 in vivo and in vitro. A specific activity of 316.8 nmol of precorrin-2 h−1·mg−1 of NirE was found for the conversion of uroporphyrinogen III to precorrin-2. At high enzyme concentrations NirE catalyzed an overmethylation of uroporphyrinogen III, resulting in the formation of trimethylpyrrocorphin. Substrate inhibition was observed at uroporphyrinogen III concentrations above 17 μm. The protein did bind SAM, although not with the same avidity as reported for other SAM-dependent uroporphyrinogen III methyltransferases involved in siroheme and cobalamin biosynthesis. A P. aeruginosa nirE transposon mutant was not complemented by native cobA encoding the SAM-dependent uroporphyrinogen III methyltransferase involved in cobalamin formation. However, bacterial growth of the nirE mutant was observed when cobA was constitutively expressed by a complementing plasmid, underscoring the special requirement of NirE for heme d1 biosynthesis. [ABSTRACT FROM AUTHOR]

Objective: Adrenergic stimulation of the heart leads to activation of the phospholipase D signal transduction pathway with formation of the intracellular second messengers phosphatidic acid and diacylglycerol, which may play a role in the development of myocardial hypertrophy by activating mitogen-activated protein kinases and protein kinase C. So far, the adrenergic receptor subtypes mediating activation of cardiac phospholipase D are not known. Methods: We developed an assay for determination of phospholipase D activity in the isolated perfused rat heart. Utilizing the phospholipase D specific transphosphatidylation reaction the stable product phosphatidylethanol (PEtOH) is formed in rat hearts perfused in the presence of 1% ethanol. Myocardial PEtOH formation was used as a marker of phospholipase D activity and was determined by HPLC and evaporative light-scattering detection (PEtOH μg/mg myocardial protein). Results: Basal PEtOH formation in unstimulated hearts was 0.06±0.01 μg/mg. Stimulation of the hearts with norepinephrine resulted in a concentration-dependent phospholipase D activation with a maximum formation of PEtOH (0.17±0.01 μg/mg) at 100 μmol/l norepinephrine. The norepinephrine-induced increase in PLD activity was completely blocked by the α1-adrenoceptor antagonist prazosin and was unaffected by the β-adrenoceptor antagonist propranolol. Further characterisation of α1-adrenoceptor subtypes with selective α1-adrenoceptor antagonists demonstrated a complete inhibition of the norepinephrine-induced phospholipase D activation by WB 4101 (α1A-selective: 0.06±0.01 μg/mg) and by BMY 7378 (α1D-selective: 0.07±0.01 μg/mg). In contrast, the α1B-adrenoceptor antagonist chloroethylclonidine had no inhibitory effect on norepinephrine-stimulated phospholipase D activity (0.14±0.01 μg/mg). Conclusion: Adrenergic activation of the cardiac phospholipase D signal transduction pathway is mediated by α1-adrenoceptors. Here, the α1A-adrenoceptor subtype, but not the α1B-adrenoceptor are coupled to activation of cardiac phospholipase D. [Copyright &y& Elsevier]