Some properties of formate dehydrogenase,accumulation and incorporation of 185W-tungsten into proteins of Clostridium formicoaceticum

Formate dehydrogenase of Clostridium formicoaceticum used only methyl and benzyl viologen, but not NAD as electron acceptor. The S_0.5 values were 0.9×10^-4 M for formate and 5.8×10^-3 M for methyl viologen. Using potassium phosphate buffer a pH-optimum of 7.9 was observed. The initial velocity of the formate dehydrogenase activity reached a maximum at 70°C, whereas the activity was stable only up to 50°C. The level of formate dehydrogenase in C. formicoaceticum was increased to its maximum when 10^-6 M selenite and 10^-7 M tungstate were added to a synthetic medium. Addition of molybdate instead of tungstate did not increase the level of formate dehydrogenase. ¹⁸⁵W-tungsten was concentrated about 100-fold by C. formicoaceticum; molybdate had no major effect on the uptake of tungsten. ¹⁸⁵W-tungsten was found almost exclusively in the soluble fluid and was predominantly recovered after chromatography in a protein of about 88000 molecular weight. Occasionally a labelled protein of low molecular weight was observed. Again molybdate added even in high molar excess did not influence the labelling pattern. No radioactivity peak could be obtained at the elution peak of formate dehydrogenase activity. The extreme instability of formate dehydrogenase prevented further purification.Abbreviations FDH formate dehydrogenase - DTE dithioerythritol - HEPES hydroxyethylpiperazine N-2-ethane sulconic acid - TEA triethylamine - DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulfate - TTC triphenyltetrazolium     

Differentiation between Clostridium acidiurici and Clostridium cylindrosporum on the basis of specific metal requirements for formate dehydrogenase formation

The formate dehydrogenases of Clostridium acidiurici and of C. cylindrosporum coupled the oxidation of formate with the reduction of viologen dyes. The basal activity level was about 0.85 moles/min s mg of protein for both species. The level of formate dehydrogenase of C. acidiurici increased 12-fold when 10^-7 M tungstate and selenite were present during growth. Molybdate exerted no effect. On the other hand, molybdate and selenite were required to increase the formate dehydrogenase of C. cylindrosporum, and tungstate exhibited an antagonistic effect in this organism.Growth on hypoxanthine generally depended on the addition of bicarbonate. Supplementation with tungstate and selenite accelerated growth of C. acidiurici and increased again the level of formate dehydrogenase. The addition of both, molybdate and selenite was necessary to initiate growth of C. cylindrosporum and to form an active formate dehydrogenase.The differences in the requirement for metal ion supplementation to form high levels of formate dehydrogenase and their involvement in hypoxanthine degradation can be used to differentiate between C. acidiurici and C. cylindrosporum.Abbreviation FDH formate dehydrogenase     

Expression of multidrug transporter P-glycoprotein in Pichia pastoris affects the host's methanol metabolism

Pichia pastoris KM71H (Mut^S) is an efficient producer of hard-to-express proteins such as the membrane protein P-glycoprotein (Pgp), an ATP-powered efflux pump which is expressed properly, but at very low concentration, using the conventional induction strategy. Evaluation of different induction strategies indicated that it was possible to increase Pgp expression by inducing the culture with 20% media containing 2.5% methanol. By quantifying methanol, formaldehyde, hydrogen peroxide and formate, and by measuring alcohol oxidase, catalase, formaldehyde dehydrogenase, formate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenases, it was possible to correlate Pgp expression to the induction strategy. Inducing the culture by adding methanol with fresh media was associated with decreases in formaldehyde and hydrogen peroxide, and increases in formaldehyde dehydrogenase, formate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenases. At these conditions, Pgp expression was 1400-fold higher, an indication that Pgp expression is affected by increases in formaldehyde and hydrogen peroxide. It is possible that Pgp is responsible for this behaviour, since the increased metabolite concentrations and decreased enzymatic activities were not observed when parental Pichia was subjected to the same growth conditions. This report adds information on methanol metabolism during expression of Pgp from P. pastoris Mut^S strain and suggests an expression procedure for hard-to-express proteins from P. pastoris.     

The Co-immobilization of NAD and Dehydrogenases and Its Application to Bioreactors for Synthesis and Analysis

A polymerizable NAD derivative, N⁶-[N-[N-(2-hydroxy-3-methacrylamidopropyl)carbamoylmethyl]carbamoylmethyl]-NAD, formate dehydrogenase, and malate dehydrogenase were entrapped all together in polyacrylamide gels. The entrapment was carried out by radical copolymerization, and consequently NAD was bound on the matrix which enclosed the enzymes. These gels had the function of producing l-malate from oxalacetate and formate. The l-malate production was also continuously done in a column reactor for 3 days. Another gel was similarly prepared with N⁶-[N-(6-methacrylamidohexyl)carbamoylmethyl]-NAD, horse liver alcohol dehydrogenase, and diaphorase. This gel was shown to catalyze the formation of resorufin from resazurin and ethanol. This gel was applicable to ethanol analysis using a fluorescence spectrophotometer to determine resorufin. The analyzer was usable for one week.     

Localization of the enzymes involved in H2 and formate metabolism inSyntrophospora bryantii

Cell-free extracts of crotonate-grown cells of the syntrophic butyrate-oxidizing bacteriumSyntrophospora bryantii contained high hydrogenase activities (8.5–75.8 µmol · min^–1 mg^–1 protein) and relatively low formate dehydrogenase activities (0.04–0.07 µmol · min^–1 mg^–1 protein). The K _M value and threshold value of the hydrogenase for H₂ were 0.21 mM and 18 µM, respectively, whereas the K _M value and threshold value of the formate dehydrogenase for formate were 0.22 mM and 10 µM, respectively. Hydrogenase, butyryl-CoA dehydrogenase and 3-OH-butyryl-CoA dehydrogenase were detected in the cytoplasmic fraction. Formate dehydrogenase and CO₂ reductase were membrane-bound, likely located at the outer aspect of the cytoplasmic membrane. Results suggest that during syntrophic butyrate oxidation H₂ is formed intracellularly while formate is formed at the outside of the cell.     

Induction and regulation of ribulose bisphosphate carboxylase activity in Rhizobium japonicum during formate-dependent growth

Rhizobium japonicum CJ1 was capable of growing using formate as the sole source of carbon and energy. During aerobic growth on formate a cytoplasmic NAD⁺-dependent formate dehydrogenase and ribulose bisphosphate carboxylase activity was demonstrated in cell-free extracts, but hydrogenase enzyme activity could not be detected. Under microaerobic growth conditions either formate or hydrogen metabolism could separately or together support ribulose bisphosphate carboxylase-dependent CO₂ fixation. A number of R. japonicum strains defective in hydrogen uptake activity were shown to metabolise formate and induce ribulose bisphosphate carboxylase activity. The induction and regulation of ribulose bisphosphate carboxylase is discussed.Abbreviations hup hydrogen uptake - MOPS 3-(N-morpholino)-propanesulphonate - TSA tryptone soya agar - RuBP ribulose 1,5-bisphosphate - FDH formate dehydrogenase     

Enantioselective reduction of carbonyl compounds by whole-cell biotransformation, combining a formate dehydrogenase and a (R)-specific alcohol dehydrogenase

A whole-cell biotransformation system for the reduction of prochiral carbonyl compounds, such as methyl acetoacetate, to chiral hydroxy acid derivatives [methyl (R)-3-hydroxy butanoate] was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. Alcohol dehydrogenase from Lactobacillus brevis catalyzes a highly regioselective and enantioselective reduction of several ketones or keto acid derivatives to chiral alcohols or hydroxy acid esters. The adh gene encoding for the alcohol dehydrogenase of L. brevis was expressed in E. coli. As expected, whole cells of the recombinant strain produced only low quantities of methyl (R)-3-hydroxy butanoate from the substrate methyl acetoacetate. Therefore, the fdh gene from Mycobacterium vaccae N10, encoding NAD⁺-dependent formate dehydrogenase, was functionally coexpressed. The resulting two-fold recombinant strain exhibited an in vitro catalytic alcohol dehydrogenase activity of 6.5 units mg^–1 protein in reducing methyl acetoacetate to methyl (R)-3-hydroxy butanoate with NADPH as the cofactor and 0.7 units mg^–1 protein with NADH. The in vitro formate dehydrogenase activity was 1.3 units mg^–1 protein. Whole resting cells of this strain catalyzed the formation of 40 mM methyl (R)-3-hydroxy butanoate from methyl acetoacetate. The product yield was 100 mol% at a productivity of 200 mol g^–1 (cell dry weight) min^–1. In the presence of formate, the intracellular [NADH]/[NAD⁺] ratio of the cells increased seven-fold. Thus, the functional overexpression of alcohol dehydrogenase in the presence of formate dehydrogenase was sufficient to enable and sustain the desired reduction reaction via the relatively low specific activity of alcohol dehydrogenase with NADH, instead of NADPH, as a cofactor.     

Nicotinamide adenine dinucleotide-dependent formate dehydrogenase from Rhodopseudomonas palustris

Summary Formate dehydrogenase in extracts of the facultative phototroph, Rhodopseudomonas palustris was shown to be soluble and NAD-linked. The flavin nucleotides, FMN and FAD, stimulated the rate of NAD reduction about fourfold. Reduction of artificial electron acceptors such as DCPIP and cytochrome c was also stimulated by FMN and FAD. The pH optimum for the reduction of NAD was pH 8.0, in contrast to pH 6.8 for cytochrome c and DCPIP reduction. The apparent K _m for formate as measured by NAD reduction was 2.6×10^-4 M. Although the addition of thiosulfate or yeast extract to the formate medium increased both the growth rate and yield of Rhps. palustris, they had little effect on the activity of formate dehydrogenase.     

<Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> NAD-dependent, <Emphasis Type="SmallCaps">D</Emphasis>-fructose reducing,polyol dehydrogenases activity: screening,medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l^–1, 7.2 g peptone l–1 and 1.8 g yeast extract l^–1. Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂.     

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 M, 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.     

S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K _mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K _mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K _mof 40 mM) but a relatively high affinity for S-formylglutathione (K _mof 1.1 mM). The K _mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.     

Characterization of a thermally stable and organic solvent-adaptative NAD+ -dependent formate dehydrogenase from Bacillus sp. F1

Aims: To characterize a robust NAD⁺‐dependent formate dehydrogenase firstly obtained from a nonmethylotroph, Bacillus sp. F1. Methods and Results: The Bacillus sp. F1 NAD⁺‐dependent formate dehydrogenase (BacFDH) gene was cloned by TAIL‐PCR and heterologous expressed in Escherichia coli. BacFDH was stable at temperatures below 55°C, and the half‐life at 60°C was determined as 52·9 min. This enzyme also showed a broad pH stability and retained more than 80% of the activities after incubating in buffers with different pH ranging from 4·5 to 10·5 for 1 h. The activity of BacFDH was significantly enhanced by some metal ions. Moreover, BacFDH exhibited high tolerance to 20% dimethyl sulfoxide, 60% acetone, 10% methanol, 20% ethanol, 60% isopropanol and 20% n‐hexane. Like other FDHs, BacFDH displayed strict substrate specificity for formate. Conclusion: We isolated a robust formate dehydrogenase, designated as BacFDH, which showed excellent thermal stability, organic solvent stability and a broad pH stability. Significance and Impact of the Study: The multi‐aspect stability makes BacFDH a competitive candidate for coenzyme regeneration in practical applications of chiral chemicals and pharmaceuticals synthesis with a relatively low cost, especially for the catalysis performed in extreme pH conditions and organic solvents.     

Induced synthesis of formyltetrahydrofolate synthetase in Micrococcus aerogenes

Summary The levels of formyltetrahydrofolate synthetase and cyclohydrolase in M. aerogenes were enhanced 3-to 10-fold by growth in media containing formate of histidine. This induced synthesis was decreased by the simultaneous addition of ribosides or ribotides. Histidine, but not formate, also induced the synthesis of formimino transferase and/or cyclodeaminase. The specific activities of N¹⁰-formyltetrahydrofolate deacylase, serine hydroxymethylase and N⁵, N¹⁰-methylenetetrahydrofolate dehydrogenase were not affected by formate or histidine. These observations have been discussed with respect to the known mechanisms of regulation of tetrahydrofolate linked enzymes.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday.Recipient of Research Career Award GM-K6-422.     

Oxidation of Methanol by the Yeast Pichia pastoris. Purification and Properties of the Formate Dehydrogenase

The formate dehydrogenase from the yeast Pichia pastoris IFP 206 was purified to homogeneity. The protein showed a molecular weight of 68,000 daltons and was composed of two identical subunits. Its amino acid composition was similar to those of other formate dehydrogenases and was characterized by a high content of acidic residues. The N-terminal end of the molecule was probably blocked.

The enzyme activity was NAD⁺ dependent (NADP⁺ could not replace NAD⁺). Its optimum temperature was 47°C and the activation energy 10.8 kcal/mol. The enzyme was active from pH 3.5 to 10.5 with a maximum at pH 7.5. The Michaelis constant for NAD+ and formate were respectively 0.27 and 15mM. The purified enzyme had no S-formylglutathione hydrolase activity, strongly suggesting that the true substrate was formate. NADH, cyanide and azide were strong inhibitors of the enzyme.     

Bacterial degradation of p-methoxybenzoic acid

Summary A cell-free system from a Pseudomonas sp., strain PM3, catalysed the oxidative demethylation, hydroxylation and subsequent ring cleavage of p-methoxybenzoate. Demethylation, to yield p-hydroxybenzoate, involved absorption of 1.0 mole of oxygen/mole of p-methoxybenzoate, and required reduced pyridine nucleotide (either NADH or NADPH) as cofactor. p-Hydroxybenzoate was hydroxylated to yield protocatechuate with the absorption of 1 mole of oxygen/mole of substrate, and required NADPH as cofactor. Protocatechuate was oxidized, with absorption of 1 mole of oxygen/mole of substrate, to 3-oxoadipate. The methyl group of p-methoxybenzoate was removed as formaldehyde, and oxidized to formate and carbon dioxide by formaldehyde dehydrogenase, which required GSH and NAD⁺, and formate dehydrogenase, which required NAD⁺.     

[1-14C] Acetate assimilation by obligate methylotrophs,Pseudomonas methanica and Methylosinus trichosporium

The oxidation of one carbon compounds (methane, methanol, formaldehyde, formate) and primary alcohols (ethanol, propanol, butanol) supported the assimilation of [1-¹⁴C]acetate by cell suspensions of type I obligate methylotroph; Pseudomonas methanica, Texas strain, and type II obligate methylotroph, Methylosinus trichosporium, strain PG. The amount of oxygen consumed and substrate oxidized correlated with the amount of [1-¹⁴C]acetate assimilated during oxidation of C-1 compounds and primary alcohols.Oxidation of methanol, formaldehyde, and primary alcohols in extracts of Pseudomonas methanica, Texas strain, and Methylosinus trichosporium, strain PG, was catalyzed by a phenazine methosulfate linked, ammonium ion dependent methanol dehydrogenase. The oxidation of aldehydes was catalyzed by a phenazine methosulfate linked, ammonium ion independent aldehyde dehydrogenase. Formate was oxidized by a NAD⁺ linked formate dehydrogenase.Deceased.This work was supported by Grant GB 8173 from the National Science Foundation and by a grant from the Robert A. Welch Foundation.     

Effect of dilution rate and carbon availability on Bifidobacterium breve fermentation

Bifidobacterium breve NCFB 2257 was grown in glucose-limited and nitrogen (N)-limited chemostats at dilution rates (D) from 0.04 to 0.60 h^–1, to study the effect of nutrient availability on carbohydrate metabolism. The results showed that D had little effect on fermentation product formation, irrespective of the form of nutrient limitation. However, marked differeces were observed in the distribution of fermentation products, that were attributable to glucose availability. In glucose-limited cultures, formate and acetate were the principal end-products of metabolism. Lactate was never detected under these growth conditions. In contrast, lactate and acetate were mainly formed when glucose was in excess, and formate was not produced. These results are explained by the metabolic fate of pyruvate, which can be dissimilated by either phosphoroclastic cleavage to acetyl phosphate and formate, or alternatively, it may be reduced to lactate. Enzymic studies were made to establish the mechanisms that regulated pyruvate metabolism. The data demonstrated that control was not exercised through regulation of the synthesis and activity of lactate dehydrogenase (LDH), phosphofructokinase or alcohol dehydrogenase. It is possible however, that there was competition for pyruvate by LDH and the phosphoroclastic enzyme, which would determine the levels of lactate and formate produced respectively. These results demonstrate the metabolic flexibility of B. breve, which preferentially uses lactate as an electron sink during N-limited growth, whereas under energy-limitation, carbon flow is directed towards acetyl phosphate to maximise ATP synthesis. Correspondence to: B. A. Degnan     

Formate Dehydrogenase of Clostridium thermoaceticum: Incorporation of Selenium-75, and the Effects of Selenite, Molybdate, and Tungstate on the Enzyme

The formation of the nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase in Clostridium thermoaceticum is stimulated by the presence of molybdate and selenite in the growth medium. The highest formate dehydrogenase activity was obtained with 2.5 × 10⁻⁴ M Na₂MoO₄ and 5 × 10⁻⁵ Na₂SeO₃. Tungstate but not vanadate could replace molybdate and stimulate the formation of formate dehydrogenase. Tungstate stimulated activity more than molybdate, and in combination with molybdate the stimulation of formation of formate dehydrogenase was additive. Formate dehydrogenase was isolated from cells grown in the presence of Na₂⁷⁵SeO₂, and a correlation was observed between bound ⁷⁵Se and enzyme activity.     

Biochemical and genetic studies with nitrate reductase C-gene mutants of Escherichia coli

Summary Twenty-eight narC (chlC) mutants of Escherichia coli were isolated and characterised by their resistance to chlorate, inability to use nitrate as terminal electron acceptor and positive gas reaction. The extent of gas production by the majority of mutants was almost normal but quantitative differences ranging from 40 to 100% of wild-type activity were found. Biochemical studies showed that all the mutants lacked nitrate reductase, decreasing gas production was correlated with a simultaneous decrease in formate dehydrogenase activity and the lowest gas production was due to deficiencies in formate dehydrogenase and hydrogenase. The position of narC relative to other loci was determined as: purB ... hemA ... narC ... supIII,C ... galU ... att80 ... tonB ... trp ... cysB by transduction analysis, and the mutant sites of 6 strains representing the complete range of gas reactions were clustered at this position. It is suggested that narC is the structural gene for nitrate reductase and the variations in phenotype may be due to polarity effects on neighbouring genes specifying components of the formate hydrogenlyase system. Transduction of narC by 80 could not be detected but an effect of galU ^– on phage P1kc susceptibility was demonstrated.     

Effect of tungsten and molybdenum on growth of a syntrophic coculture of <Emphasis Type="Italic">Syntrophobacter fumaroxidans</Emphasis> and <Emphasis Type="Italic">Methanospirillum hungatei</Emphasis>

The effect of tungsten (W) and molybdenum (Mo) on the growth of Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied in syntrophic cultures and the pure cultures of both the organisms. Cells that were grown syntropically were separated by Percoll density centrifugation. Measurement of hydrogenase and formate dehydrogenase levels in cell extracts of syntrophically grown cells correlated with the methane formation rates in the co-cultures. The effect of W and Mo on the activity of formate dehydrogenase was considerable in both the organisms, whereas hydrogenase activity remained relatively constant. Depletion of tungsten and/or molybdenum, however, did not affect the growth of the pure culture of S. fumaroxidans on propionate plus fumarate significantly, although the specific activities of hydrogenase and especially formate dehydrogenase were influenced by the absence of Mo and W. This indicates that the organism has a low W or Mo requirement under these conditions. Growth of M. hungatei on either formate or H₂/CO₂ required tungsten, and molybdenum could replace tungsten to some extent. Our results suggest a more prominent role for H₂ as electron carrier in the syntrophic conversion of propionate, when the essential trace metals W and Mo for the functioning of formate dehydrogenase are depleted.