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Summary In Paracoccusdenitrificans the aa3‐type cytochrome c oxidase and the bb3‐type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOQP, and demonstrate that it encodes an alternative cbb3‐type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen‐sensing mechanism. This view is corroborated by the observation that the ccoNOQP gene cluster is preceded by a gene that encodes an FNR homologue and that its promoter region contains an FNR‐binding motif. Biochemical and physiological analyses of a set of oxidase mutants revealed that, at least under the conditions tested, cytochromes aa3, bb3. and cbb3 make up the complete set of terminal oxidases in P. denitrificans. Proton‐translocation measurements of these oxidase mutants indicate that all three oxidase types have the capacity to pump protons. Previously, however, we have reported decreased H+/e coupling efficiencies of the cbb3‐type

The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu wenhoek 66, 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, although at a lower specific growth rate and with lower NOR activity compared with the wild-type strain.

Downstream of flhA, the Paracoccus denitrificans gene encoding glutathione-dependent formaldehyde dehydrogenase, an open reading frame was identified and called fghA. The gene product of fghA showed appreciable similarity with human esterase D and with the deduced amino acid sequences of open reading frames found in Escherichia coli, Haemophilus influenzae, and Saccharomyces cerevisiae. Mutating fghA strongly reduced S-formylglutathione hydrolase activity. The mutant was unable to grow on methanol and methylamine, indicating that the enzyme is essential for methylotrophic growth. S-Formylglutathione hydrolase appears to be part of a formaldehyde detoxification pathway that is universal in nature.In Paracoccus denitrificans, the oxidation of methanol, methylamine, and choline leads to the transient synthesis of formaldehyde. This poses a special regulation problem, as the concentration of this toxic compound must be kept within the limits imposed by its toxicity on the one hand and the concentration required for rapid further oxidation on the other hand. The oxidation of methanol and methylamine is catalyzed by the periplasmic quinoproteins methanol dehydrogenase and methylamine dehydrogenase, respectively (11). Formaldehyde formed during these reactions is transported to the cytoplasm by a protein-mediated mechanism (14). Choline is oxidized in several steps to glycine. In this process, formaldehyde is released in the cytoplasm. In the cytoplasm, formaldehyde is coupled to reduced glutathione (GSH) to yield S-hydroxymethyl-glutathione. The latter compound is oxidized by NADdependent formaldehyde dehydrogenase (GD-FALDH) to S-formylglutathione (27). The P. denitrificans gene (flhA) encoding this enzyme has been isolated and sequenced (21). An flhA mutant was unable to grow on methanol, methylamine, or choline, indicating that GD-FALDH is essential for methylotrophic growth of P. denitrificans. S-formylglutathione is hydrolyzed to formate and GSH by S-formylglutathione hydrolase (FGH). This enzyme has not yet been isolated from P. denitrificans, but FGH activity has been demonstrated in human tissues (26) and in the methylotrophic yeasts Candida boidinii (20) and Kloeckera sp. strain No2201 (13). FGH isolated from human liver and Kloeckera sp. strain No2201 is a homodimer with a molecular mass of 58 kDa. The C. boidinii FGH is a heterodimer with subunits of 35 and 25 kDa. In these organisms, genes encoding FGH have not been identified. Studies on the polymorphism of FGH in human erythrocytes revealed that the enzyme is identical to human esterase D (7). Human esterase D is a member of a group of nonspecific esterases. The native enzyme has a molecular weight of 70,000 and consists of two identical subunits (16). Esterase D has been found in most human tissues, but the highest activities were found in placenta, kidney, liver, and erythrocytes. The gene encoding this protein has been isolated and sequenced (15).The capacity to detoxify formaldehyde is an important feature for every organism. Both GD-FALDH and FGH ar...

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Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding subunit I of the aa3-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (delta ctaDI, delta ctaDII), indicating that they are isoforms of subunit I of the aa3-type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. This protohaem-containing oxidase, called cytochrome bb3, is the only quinol oxidase expressed under the conditions used. In a triple oxidase mutant (delta ctaDI, delta ctaDII, cyoB::KmR) an alternative cytochrome c oxidase has been characterized; this cbb3-type oxidase has been partially purified. Both cytochrome aa3 and cytochrome bb3 are redox-driven proton pumps. The proton-pumping capacity of cytochrome cbb3 has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.

NAD-and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) ofParacoccus denitrificans has been purified as a tetramer with a relative molecular mass of 150 kDa. The gene encoding GD-FALDH (flhA) has been isolated, sequenced, and mutated by insertion of a kanamycin resistance gene. The mutant strain is not able to grow on methanol, methylamine, or choline, while heterotrophic growth is not influenced by the mutation. This finding indicates that GD-FALDH of P. denitrificans is essential for the oxidation of formaldehyde produced during methylotrophic growth.Paracoccus denitrificans is a gram-negative, aerobic soil bacterium which is able to grow on methanol, methylamine, and choline. The oxidation of methanol and methylamine to formaldehyde is catalyzed by the periplasmically located enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively. During growth on methylamine, formaldehyde is transported to the cytoplasm by a transport mechanism in which a transport protein is involved (16). During the oxidation of choline to glycine, several molecules of formaldehyde are produced. In the presence of formaldehyde and reduced glutathione, the compound S-hydroxymethylglutathione is nonenzymatically formed. NAD-and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) oxidizes S-hydroxymethylglutathione to S-formylglutathione (14,19,20,24,27), which is oxidized further via formate to carbon dioxide. In this report, we describe the purification of GD-FALDH of P. denitrificans and the isolation and mutagenesis of the gene encoding this enzyme. From growth characteristics of the mutant strain, it can be concluded that GD-FALDH of P. denitrificans is essential for methylotrophic growth.Purification and biochemical analysis of GD-FALDH. GD-FALDH was purified approximately 50-fold from methylamine-grown P. denitrificans cells, as shown in Table 1. By this method, 65 g (wet weight) of cells was harvested and washed with 50 mM Tris hydrochloride (pH 7.5). The cells were disrupted with a French pressure cell, yielding the cell extract. Enzyme activity was measured routinely by determining the rate of NADH formation at 340 nm at room temperature. The assay mixture contained (final concentrations) 0.1 M Na 4 P 2 O 7 -HCl (pH 9.0), 2 mM (reduced) glutathione, and 2.5 mM NAD. After 30 s of incubation with enzyme solution, the reaction was started by adding formaldehyde to a final concentration of 5 mM. Activities were calculated by using a molar absorption coefficient for NADH at 340 nm of 6,220 M Ϫ1 cm Ϫ1 (4).(NH 4 )SO 4 was added to the cell extract. The enzyme precipitated between 25 and 60% saturation. The precipitate was dissolved in 10 mM potassium phosphate buffer (KPB; pH 7.0) and applied to a Phenyl-Sepharose HP column (12.4 by 2.6 cm) equilibrated with 1.5 M (NH 4 )SO 4 in 10 mM KPB (pH 7.0). After washing of the column with the same buffer, elution occurred with a gradient of 1.5 to 0 M (NH 4 )SO 4 in 10 mM KPB in 3 h at a flow rate 3 ml/min. GD-FALDH eluted when the (NH 4 )SO 4 concentration ...

The nit and nor genes, which encode nitrite and nitric oxide reductase, lie close together on the DNA of Paracoccus denitrificans. We here identify an adjacent gene, nnr, which is involved in the expression of nit and nor under anaerobic conditions. The corresponding protein of 224 amino acids is homologous with the family of FNR proteins, although it lacks the N-terminal cysteines. A mutation in the nnr gene had a negative effect on the expression of nitrite and nitric oxide reductase. Synthesis of membrane bound nitrate reductase, of nitrous oxide reductase, and of the cbb3-type cytochrome c oxidase were not affected by mutation of this gene. These results suggest that denitrifieation in P. denitrificans may be governed by a signal transduction network that is similar to that involved in oxygen regulation of nitrogen metabolism in other organisms.