Isolation, sequencing, and mutagenesis of the gene encoding NAD- and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) from Paracoccus denitrificans, in which GD-FALDH is essential for methylotrophic growth

Abstract

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 ...

Modulation of Nitrosative Stress byS-Nitrosoglutathione Reductase Is Critical for Thermotolerance and Plant Growth inArabidopsis

“…It is clear that GSNOR exhibits FALDH activity in vivo in plants and other organisms. The FALDH enzyme in the aerobic soil bacterium Paracoccus denitrificans is critical for methyltrophic growth (Ras et al, 1995). A yeast FALDH deletion mutant (sfa1) showed impaired growth in the presence of formaldehyde (Fernandez et al, 1999;Achkor et al, 2003), and in Arabidopsis, overexpression of the FALDH gene (GSNOR) conferred a high resistance to formaldehyde .…”

Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron This paper is dedicated to the memory of Dr M. A. Vides, Facultad de Ciencias Quı́micas, Universidad Nacional de Córdoba, Argentina, who was a great mentor and colleague. The GenBank accession number for the sequence reported in this paper is AF130307.

“…However, it was found later that this enzyme is essential in the oxidation of methanol and other methylated compounds when they are the sole carbon source in methylotrophic bacteria such as Paracoccus denitrificans (Ras et al, 1995) and the facultative phototroph Rhodobacter sphaeroides (Barber et al, 1996 ;Barber & Donohue, 1998a). It was also shown that GSH-FDH activity is involved in microbial resistance to exogenous formaldehyde (Kaulfers & Wollman, 1988 ;Sasnauskas et al, 1992).…”

“…D21201). The alignment of these predicted amino acid sequences showed that the A. baumannii GSH-FDH enzyme contains several conserved amino acids required for the structure and enzymic activity of this protein, such as the C-40, H-62 and C-169 ligands to the active Zn, the cysteine residues at positions 92, 95, 98 and 106 that bind to the second Zn atom, and the typical NAD + -binding site 193-GLGGIG-200 (Ras et al, 1995 ;Whener et al, 1993).…”

Reversed electron transfer through the bc1complex enables a cytochrome c oxidase mutant (Δaa3/cbb3) of Paracoccus denitrificans to grow on methylamine

“…via formaldehyde and formate, to carbon dioxide. Both the oxidation of formaldehyde and formate yields one molecule of NADH [1,12].…”