Dye-linked Formaldehyde Dehydrogenase Research Articles

A fast method for the quantification of methylamine in fermentation broths by gas chromatography

The objective of this study was to develop a method for the quantitative analysis of the methylamine concentration in fermentation broths of Hyphomicrobium zavarzinii ZV 580 cultures. For this purpose an established method for the quantification of free amino acids in such matrices was adapted and validated. The detection limit was 10 μM, the calibration curve showed good linearity ( R 2 = 0.9998) in the concentration range between 0.1 and 8 mM. The standard deviation of the injection-to-injection reproducibility ( n = 10) of the retention coefficient was <1%, that of the peak area <5%. In case of the sample-to-sample reproducibility ( n = 8), the standard deviation was <5% for the retention coefficient and <10% for the peak area. The validated method was successfully applied for monitoring a fed-batch bioprocess (starting volume: 8 L, initial methylamine hydrochloride concentration: 10 mM) producing a dye-linked formaldehyde dehydrogenase in H. zavarzinii ZV 580.

Development of a fed-batch process for the production of a dye-linked formaldehyde dehydrogenase in Hyphomicrobium zavarzinii ZV 580

The dye-linked formaldehyde dehydrogenase (dlFalDH) from Hyphomicrobium zavarzinii ZV 580 processes formaldehyde in a highly selective manner and without need for NAD(P). The enzyme thus has considerable potential for technical applications if the difficulties associated with its efficient production can be resolved. In this contribution, a fed-batch bioprocess is developed, which improves both the biomass production of H. zavarzinii ZV 580 (from 0.6 to 2 g l(-1) dry mass) and the specific dlFalDH production (from 0.1 to 0.3 units g(-1) biomass), resulting in an overall improvement of the productivity by more than an order of magnitude compared to the previously reported process (Klein et al., Biochem J 301:289-295, 1994). In particular, the process uses an automated feeding strategy controlled via the dissolved oxygen concentration. In addition, our results show that the growth of H. zavarzinii ZV 580 is rather sensitive toward increasing salt concentration in the culture medium. Growth is also inhibited by the presence of surfactant-based antifoam reagents. Adjustment of the pH via the addition of methylamine instead of NaOH, on the other hand, leads to an increase in biomass yield.

Relations and functions of dye-linked formaldehyde dehydrogenase from Hyphomicrobium zavarzinii revealed by sequence determination and analysis.

faoA, the gene of the dye-linked NAD(P)-independent quinone-containing formaldehyde dehydrogenase of methylamine-grown Hyphomicrobium zavarzinii strain ZV 580 was sequenced and analyzed together with an apparent promoter region and adjoining genes in a 7.2-kb fragment of hyphomicrobial DNA. The formaldehyde dehydrogenase, identified as a periplasmic enzyme by its signal sequence, is distantly related to the soluble pyrroloquinoline-quinone-dependent glucose dehydrogenase of Acinetobacter calcoaceticus and to other predicted glucose dehydrogenase sequences. The promoter region, containing about 400 nucleotides upstream of faoA, comprised potential binding sites identical or highly similar to known consensus sequences of the sigma factors sigma(70) (housekeeping), sigma(H) (heat shock), sigma(F) (flagellar) and sigma(N) (nitrogen). The complex regulation of the transcription of faoA, which is suggested by this setting and emphasized by a possible heat-shock promoter, supports a hypothesis proposing an auxiliary role of the enzyme in lowering detrimental elevated concentrations of formaldehyde, which might arise in the course of stress or regulatory transitions disturbing balanced C(1) metabolism.

Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria.

Methylotrophic bacteria can grow on a number of substrates as energy source with only one carbon atom, such as methanol, methane, methylamine, and dichloromethane. These compounds are metabolized via the cytotoxin formaldehyde. The formaldehyde consumption pathways, especially the pathways for the oxidation of formaldehyde to CO(2) for energy metabolism, are a central and critical part of the metabolism of these aerobic bacteria. Principally, two main types of pathways for the conversion of formaldehyde to CO(2) have been described: (1) a cyclic pathway initiated by the condensation of formaldehyde with ribulose monophosphate, and (2) distinct linear pathways that involve a dye-linked formaldehyde dehydrogenase or C(1) unit conversion bound to the cofactors tetrahydrofolate (H(4)F), tetrahydromethanopterin (H(4)MPT), glutathione (GSH), or mycothiol (MySH). The pathways involving the four cofactors have in common the following sequence of events: the spontaneous or enzyme-catalyzed condensation of formaldehyde and the respective C(1) carrier, the oxidation of the cofactor-bound C(1) unit and its conversion to formate, and the oxidation of formate to CO(2). However, the H(4)MPT pathway is more complex and involves intermediates that were previously known solely from the energy metabolism of methanogenic archaea. The occurrence of the different formaldehyde oxidation pathways is not uniform among different methylotrophic bacteria. The pathways are in part also used by other organisms to provide C(1) units for biosynthetic reactions (e.g., H(4)F-dependent enzymes) or detoxification of formaldehyde (e.g., GSH-dependent enzymes).

Membrane-associated quinoprotein formaldehyde dehydrogenase from Methylococcus capsulatus Bath.

A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 micromol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)(+)-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159-167, 1999; Vorholt et al., J. Bacteriol. 180:5351-5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome b(559/569) complex as the physiological electron acceptor.

A novel dye-linked formaldehyde dehydrogenase with some properties indicating the presence of a protein-bound redox-active quinone cofactor

Dye-linked formaldehyde dehydrogenase from methylamine-grown Hyphomicrobium zavarzinii ZV 580, a tetramer of M(r) 210,000 with subunits of M(r) 54,000, was purified to homogeneity in five steps with 10% yield. The enzyme shows optimal affinity for, and activity with, formaldehyde (Km 67 microM) compared with other aldehydes. Pyridoxal phosphate, pyrroloquinoline quinone and other cofactors that would give the enzyme a distinctive absorption spectrum are absent. Slight changes are observed in the spectrum at 300-550 nm on oxidation of the enzyme with Wurster's Blue (WB) and reduction with formaldehyde. Titration of the native reduced enzyme with WB accounts for 2 mol of electrons per mol of tetrameric enzyme. The circumstantial evidence supporting the presence of a redox-active quinone cofactor bound to the polypeptide chain comprises a signal at g = 2.0049 in the X-band e.p.r. spectrum of the enzyme oxidized with WB, which disappears on reduction with formaldehyde, and a positive reaction of the native as well as the denatured and dialysed enzyme in the redox-cycling assay with glycinate and NitroBlue Tetrazolium (quinone staining). The oxidized enzyme is inhibited by equimolar amounts of phenylhydrazine, which is also a reductant. Hydrazone formation was absent with completely inhibited enzyme, according to photometric evidence. Likewise, the glycinate-dependent reduction of NitroBlue Tetrazolium was not affected by the inhibitor. It is concluded that an oxidation product of the hydrazine is the actual inhibitor which reacts with an amino acid residue of the active site rather than with the prospective quinone cofactor.

Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C1-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 μM, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol-1 at a dilution rate of 0.05 h-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C1-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.

Induction and selection of formaldehyde-based resistance inPseudomonas aeruginosa

A formaldehyde resistant (R) phenotype ofPseudomonas aeruginosa was isolated from a formaldehydesensitive (S) parent by sequential treatment with 1,3,5-tris-(ethyl)hexahydro-s-triazine (ET). The resistance of the (R) strain to treatment with ET was approximately 3-fold higher than the parental (S) strain. Two modes of resistance to ET, and simultaneous resistance to formaldehyde, are demonstrated: (1) transient or induced resistance is expressed during shor-term exposure to ET, and this resistance is gradually lost during subsequent growth in the absence of ET, and (2) resistance that results from a stable phenotypic change in the (S) strain following sequential treatment with ET ((R) strain phenotype). The observed activities of three forms of the formaldehyde oxidizing enzyme, formaldehyde dehydrogenase, are strongly correlated with the relative response of the (S) and (R) strains to treatment with ET. The observed resistance of the (R) strain appears to be due to high levels of an NAD+-linked, glutathione-dependent form of formaldehyde dehydrogenase as well as a dye-linked formaldehyde dehydrogenase. The transient or induced response of the (R) strain involves an increase in activity of the dye-linked formaldehyde dehydrogenase. The induced response of the (S) strain and an ATCC strain ofP. aeruginosa, however, is correlated with the two forms of the NAD+-linked enzyme (glutathione-dependent (EC 1.2.1.1) and independent (EC 1.2.1.46)) with no contribution from the dye-linked enzyme.

A Possible Alternative Mechanism for the Oxidation of Formaldehyde to Formate

A survey of the occurrence of a no. of enzymes possibly involved in the C1 dissimilatory pathway in organisms using the serine pathway of C assimilation indicated that neither the methanol dehydrogenase nor the NH4-independent dye-linked formaldehyde dehydrogenase had a specific role in the oxidn. of formaldehyde. Conversely, the activities of enzymes involved in the methenyl-THF pathway were induced during growth on MeOH or MeNH2, suggesting a potential dissimilatory role for this pathway. [on SciFinder (R)]

Partial Purification and Characterization of a Dye-linked Formaldehyde Dehydrogenase from Hyphomierobium X

Dye-linked formaldehyde dehydrogenase (I) was partially purified (11-fold) from MeOH- and EtOH-grown Hyphomicrobium X. Through not homogeneous, it did not contain MeOH dehydrogenase, formate dehydrogenase, or cytochrome c. Methylphenazonium methylsulfate (II), cytochrome c, Wurster's Blue, dichlorophenolindophenol (III), and methylphenazonium ethosulfate could act as primary electron acceptors with apparent Km values for the first 3 substances of 0.69, 128, and 3.07 mM, resp. The activity of partially purified I could be maintained over 3 mo at -20 Deg in the presence of EtOH (10%). The mol. wt. of I was 83,500 and it was active from pH 6.5 to 9.0 with max. activity at pH 7.2 using cytochrome c as electron acceptor and at pH 7.6 when II and III were used. The Km values for formaldehyde, acetaldehyde, propionaldehyde, and heptaldehyde as substrates were 2860, 270, 13, and 2.5 mM, resp. I is probably a general aldehyde dehydrogenase not directly involved in the dissimilation of C-1 compds. [on SciFinder (R)]

The Isolation of Pleiotropic Mutants of Pseudomonas aminovorans Deficient in the Ability to Grow on Methylamine and an Examination of their Enzymic Constitution

Summary: Mutants deficient in several enzymes of methylamine oxidation and assimilation have been obtained by treating Pseudomonas aminovorans with ultraviolet light. One of these mutants was studied in detail and was shown to lack the enzymes of trimethylamine and dimethylamine oxidation. In addition, activities of the following enzymes, all postulated as being involved in C1 metabolism, were lost: hydroxypyruvate reductase, serine-glyoxylate aminotransferase, isocitrate lyase, phosphoeno/pyruvate carboxylase, N-methylglutamate dehydrogenase, γ-glutamylmethylamide synthetase, formate dehydrogenase and dye-linked formaldehyde dehydrogenase. In contrast, three enzymes - NAD+,glutathione-linked formaldehyde dehydrogenase, N-methylalanine dehydrogenase and N-methylglutamate synthase - were retained. This phenotype suggests either a lesion in a regulatory gene, a deletion in an early structural gene of an operon or the loss of a plasmid. Results obtained indicated that P. aminovorans possesses an isocitrate lyase-serine pathway.

Synthesis of Certain Assimilatory and Dissimilatory Enzymes during Bacterial Adaptation to Growth on Trimethylamine

Summary: During the adaptation of Pseudomonas aminovorans from growth on succinate to growth on trimethylamine, the following enzymes were synthesized in the lag phase before exponential growth on trimethylamine began: trimethylamine and dimethylamine mono-oxygenases, trimethylamine-N-oxide aldolase (demethylase), glutathione- and NAD-dependent formaldehyde dehydrogenase, dye-linked formaldehyde dehydrogenase, hydroxypyruvate reductase and N-methylglutamate dehydrogenase. Differential plots suggested that the rate of enzyme synthesis in the lag phase exceeded the rate of synthesis during exponential growth. The evidence suggests that the enzymes discussed are essential for growth on trimethylamine, while the NADPH-dependent N-methylalanine dehydrogenase is not involved.