For many pathogenic bacteria like Pseudomonas aeruginosa heme is an essential source of iron. After uptake, the heme molecule is degraded by heme oxygenases to yield iron, carbon monoxide, and biliverdin. The heme oxygenase PigA is only induced under ironlimiting conditions and produces the unusual biliverdin isomers IX and IX␦. The gene for a second putative heme oxygenase in P. aeruginosa, bphO, occurs in an operon with the gene bphP encoding a bacterial phytochrome. Here we provide biochemical evidence that bphO encodes for a second heme oxygenase in P. aeruginosa. HPLC, 1 H, and 13 C NMR studies indicate that BphO is a "classic" heme oxygenase in that it produces biliverdin IX␣. The data also suggest that the overall fold of BphO is likely to be the same as that reported for other ␣-hydroxylating heme oxygenases. Recombinant BphO was shown to prefer ferredoxins or ascorbate as a source of reducing equivalents in vitro and the ratelimiting step for the oxidation of heme to biliverdin is the release of product. In eukaryotes, the release of biliverdin is driven by biliverdin reductase, the subsequent enzyme in heme catabolism. Because P. aeruginosa lacks a biliverdin reductase homologue, data are presented indicating an involvement of the bacterial phytochrome BphP in biliverdin release from BphO and possibly from PigA.
It is well documented that, in general, amino acids are used in the l-form, and enzymes involved in their metabolism are stereospecific for the l-enantiomers. However, d-amino acids are widely distributed in living organisms [1]. Examples of the natural occurrence of d-amino acids include d-amino acid-containing natural peptide toxins [2], antibacterial diastereomeric peptides [3], and the presence of d-amino acids at high concentrations in human brain [4]. In plants d-amino acids were detected in gymnosperms as well as monoand dicotyledonous angiosperms of major plant families. Free d-amino acids in the low percentage range of 0.5-3% relative to their l-enantiomers are principle constituents of plants [5]. The functions of d-amino acids and their metabolism are largely unknown. Various pyridoxal-5¢-phosphate (PLP)-dependent enzymes that catalyse elimination and replacement reactions of amino acids have been purified and characterized [6]. In several organisms d-cysteine desulfhydrase (d-CDes) activity (EC 4.1.99.4) was measured; this enzyme decomposes d-cysteine into pyruvate, H 2 S, and NH 3 . A gene encoding a putative d-CDes protein was identified in Arabidopsis thaliana (L) Heynh. based on high homology to an Escherichia coli protein called YedO that has d-CDes activity. The deduced Arabidopsis protein consists of 401 amino acids and has a molecular mass of 43.9 kDa. It contains a pyridoxal-5¢-phosphate binding site. The purified recombinant mature protein had a K m for d-cysteine of 0.25 mm. Only d-cysteine but not l-cysteine was converted by d-CDes to pyruvate, H 2 S, and NH 3 . The activity was inhibited by aminooxy acetic acid and hydroxylamine, inhibitors specific for pyridoxal-5¢-phosphate dependent proteins, at low micromolar concentrations. The protein did not exhibit 1-aminocyclopropane-1-carboxylate deaminase activity (EC 3.5.99.7) as homologous bacterial proteins. Western blot analysis of isolated organelles and localization studies using fusion constructs with the green fluorescent protein indicated an intracellular localization of the nuclear encoded d-CDes protein in the mitochondria. d-CDes RNA levels increased with proceeding development of Arabidopsis but decreased in senescent plants; d-CDes protein levels remained almost unchanged in the same plants whereas specific d-CDes activity was highest in senescent plants. In plants grown in a 12-h light ⁄ 12-h dark rhythm d-CDes RNA levels were highest in the dark, whereas protein levels and enzyme activity were lower in the dark period than in the light indicating post-translational regulation. Plants grown under low sulfate concentration showed an accumulation of d-CDes RNA and increased protein levels, the d-CDes activity was almost unchanged. Putative in vivo functions of the Arabidopsis d-CDes protein are discussed.Abbreviations
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