The formaldehyde dehydrogenase of Rhodococcus erythropolis, a trimeric enzyme requiring a cofactor and active with alcohols

During growth on compounds containing methyl groups a formaldehyde dehydrogenase is induced in the gram-positive bacteria Rhodococcus erythropolis. This formaldehyde dehydrogenase has been purified to homogeneity using affinity chromatography and permeation chromatography. The isoelectric point of the enzyme was 4.7. The molar mass of the native enzyme was determined as 130 000 g/mol. Sodium dodecyl sulfate gel electrophoresis yielded a single subunit with a molar mass of 44000 g/mol. These results, together with cross-linking experiments which yielded monomer, dimer, and trimer bands, are consistent with a trimeric subunit structure of the formaldehyde dehydrogenase. A heat-stable cofactor of low molar mass was required for activity with formaldehyde as substrate. This cofactor was found to be oxidizable, but active only in its reduced form. Preparative electrofocusing revealed that the cofactor is a weak acid with a pK of about 6.5. The enzyme was active with the homologous series of the primary alcohols, ethanol up to octanol, without requiring the presence of the cofactor. A mutant without formaldehyde dehydrogenase activity was not impaired in its growth with ethanol as substrate. It is suggested that the alcohols mimic the true substrate of the formaldehyde dehydrogenase, which could be a hydroxymethyl derivative of the cofactor, resulting from the addition of formaldehyde.     

Sequence of the gene for a NAD(P)-dependent formaldehyde dehydrogenase (class III alcohol dehydrogenase) from a marine methanotroph Methylobacter marinus A45

Abstract A fragment of Methylobacter marinus A45 DNA has been cloned and sequenced, and an open reading frame has been identified that could code for a 46-kDa polypeptide. Comparison of the deduced amino acid sequence of the polypeptide against the protein data bank has revealed strong similarity with a number of alcohol dehydrogenases, with highest similarity towards class III alcohol dehydrogenases, which recently have been shown to be identical to glutathione-dependent formaldehyde dehydrogenases. We were unable to measure appreciable levels of NAD(P)-dependent formaldehyde dehydrogenases or alcohol dehydrogenase activities using aldehydes or primary or secondary alcohols in cell-free extracts from batch cultures of M. marinus A45. However, formaldehyde dehydrogenases activity was detected on zymograms. Our data suggest that, although NAD(P)-linked formaldehyde dehydrogenase or alcohol dehydrogenase activities are undetectable in cell-free extracts of most methylotrophs employing the ribulose monophosphate pathway for formaldehyde assimilation and dissimilation, the gene encoding formaldehyde dehydrogenase is present in M. marinus A45 and may be present in more of these organisms as well.     

NAD- and co-substrate (GSH or factor)-dependent formaldehyde dehydrogenases from methylotrophic microorganisms act as a class III alcohol dehydrogenase

Abstract The methylotrophic yeasts, Hansenula polymorpha and Candida boidinii , and the methylotrophic Gram-negative bacteria, Paracoccus denitrificans and Thiobacillus versutus (but not Methylophaga marina ), contain NAD/GSH-dependent formaldehyde dehydrogenase when grown on C₁-compounds. The enzymes appeared to be similar to each other and to the mammalian counterparts with respect to substrate specificity, including the ability to act as an alcohol dehydrogenase class III. The Gram-positive bacteria, Amycolatopsis methanolica and Rhodococcus erythropolis , possess NAD/Factor-dependent formaldehyde dehydrogenase when grown on C₁-compounds or on C₁-unit-containing substrates, respectively. These enzymes also exhibit alcohol dehydrogenase class III activity. Thus, like the mammalian ones, methylotrophic formaldehyde dehydrogenases show dual substrate specificity, suggesting that this is an inherent property of the enzyme.     

Cyclopropanol in the exploration of bacterial alcohol oxidation

Abstract Cyclopropanol selectively inhibits bacterial alcohol oxidation proceeding via NAD-independent, quinoprotein alcohol dehydrogenases. Thus, for instance, alcohol oxidation by Pseudomonas aeruginosa , grown on ethanol, was inhibited for about 50% by cyclopropanol treatment. Accordingly, cell-free extracts of untreated cells had nearly equal activities of quinoprotein and NAD-dependent alcohol dehydrogenases, whereas only the latter enzyme activity was found in cell-free extracts of cyclopropanol-treated cells. Upon incubation of Hyphomicrobium X with cyclopropanol, oxidation of alcohols was blocked while formaldehyde oxidation was not. Therefore, methanol dehydrogenase in this organism is not specifically involved in formaldehyde oxidation. The examples show that cyclopropanol-derived substrates are potential tools in revealing the physiological role of bacterial alcohol dehydrogenases.     

Histone demethylase LSD1 is a folate-binding protein

Methylation of lysine residues in histones has been known to serve a regulatory role in gene expression. Although enzymatic removal of the methyl groups was discovered as early as 1973, the enzymes responsible for their removal were isolated and their mechanism of action was described only recently. The first enzyme to show such activity was LSD1, a flavin-containing enzyme that removes the methyl groups from lysines 4 and 9 of histone 3 with the generation of formaldehyde from the methyl group. This reaction is similar to the previously described demethylation reactions conducted by the enzymes dimethylglycine dehydrogenase and sarcosine dehydrogenase, in which protein-bound tetrahydrofolate serves as an accepter of the formaldehyde that is generated. We now show that nuclear extracts of HeLa cells contain LSD1 that is associated with folate. Using the method of back-scattering interferometry, we have measured the binding of various forms of folate to both full-length LSD1 and a truncated form of LSD1 in free solution. The 6R,S form of the natural pentaglutamate form of tetrahydrofolate bound with the highest affinity (K(d) = 2.8 μM) to full-length LSD1. The fact that folate participates in the enzymatic demethylation of histones provides an opportunity for this micronutrient to play a role in the epigenetic control of gene expression.     

Enzymes involved in the anoxic utilization of phenyl methyl ethers by <Emphasis Type="Italic">Desulfitobacterium hafniense</Emphasis> DCB2 and <Emphasis Type="Italic">Desulfitobacterium hafniense</Emphasis> PCE-S

Phenyl methyl ethers are utilized by Desulfitobacterium hafniense DCB2 and Desulfitobacterium hafniense PCE-S; the methyl group derived from the O-demethylation of these substrates can be used as electron donor for anaerobic fumarate respiration or dehalorespiration. The activity of all enzymes involved in the oxidation of the methyl group to carbon dioxide via the acetyl-CoA pathway was detected in cell extracts of both strains. In addition, a carbon monoxide dehydrogenase activity could be detected. Activity staining of this enzyme indicated that the enzyme is a bifunctional CO dehydrogenase/acetyl-CoA synthase.     

Alcohol Dehydrogenases: Identification and Names for Gene Families

Plant gene products that have been described as `alcohol dehydrogenases' are surveyed and related to their CPGN nomenclature. Most are Zn-dependent medium chain dehydrogenases, including `classical' alcohol dehydrogenase (Adh1), glutathione-dependent formaldehyde dehydrogenase (Fdh1), cinnamyl alcohol dehydrogenase (Cad2), and benzyl alcohol dehydrogenase (Bad1). Plant gene products belonging to the short-chain dehydrogenase class should not be called alcohol dehydrogenases unless such activity is shown.     

The mechanism of microbial resistance to hexahydro-1,3,5-triethyl-s-triazine

Summary Four strains ofPseudomonas putida and two unidentifiedPseudomonas species that were resistant to hexahydro-1,3,5-triethyl-s-triazine (HHTT) were shown to be resistant to formaldehyde as well. Conjugation experiments revealed that: (a) HHTT and formaldehyde resistance was cotransferred in every case where exconjugants were recovered; (b) in every case HHTT resistance and formaldehyde resistance were expressed to the same level in the exconjugant as in the donor; (c) resistance to either HHTT or formaldehyde alone was never observed; and (d) in instances where HHTT and formaldehyde resistance in the exconjugants was unstable, the exconjugants lost resistance to both agents simultaneously and never to one agent alone. Resistant organisms (e.g.P. putida 3-T-15²) had high levels of formaldehyde dehydrogenase and this enzyme appeared to be constitutively expressed. It was concluded that resistance to HHTT was due to resistance to its degradation product, formaldehyde, via detoxification of formaldehyde by formaldehyde dehydrogenase. HHTT- and formaldehyde-sensitive organisms had barely detectable levels (most likely repressed levels) of formaldehyde dehydrogenase. Although speculative, it is possible that formaldehyde resistance may be due to a mutation resulting in derepression of the gene coding for formaldehyde dehydrogenase. While it could not be discerned whether HHTT resistance and formaldehyde resistance were carried on two separate but closely linked genes or if only one gene was involved, the evidence suggested that only one gene was involved. Similarly, it could not be determined whether HHTT and formaldehyde resistance was encoded by chromosomal or plasmid genes.     

Initial microbial adhesion is a determinant for the strength of biofilm adhesion

Abstract A considerable amount of methylformate accumulated in the culture medium of methanol-grown methylotrophic yeasts. Methylformate is considered as an intermediate in a novel formaldehyde oxidation pathway. Through investigations with Pichia methanolica , methylformate formation was found to be catalysed by a new type of alcohol dehydrogenase, which was named methylformate synthase. When cells were grown on a relatively high concentration of methanol or exposed to a high concentration of formaldehyde, formation of methylformate was enhanced and the level of methylformate synthase in the cells increased. How methylformate synthase is involved in formaldehyde oxidation and formaldehyde detoxification is discussed.     

Methanol dissimilation in Xanthobacter H4-14: activities, induction and comparison to Pseudomonas AM1 and Paracoccus denitrificans

Methanol dissimilatory enzymes detected in the methanol autotroph Xanthobacter H4-14 were a typical phenazine methosulphate-linked methanol dehydrogenase, a NAD+-linked formate dehydrogenase, and a dye-linked formaldehyde dehydrogenase that could be assayed only by activity stains of polyacrylamide gels. This same methanol dehydrogenase activity was found in ethanol-grown cells and was apparently utilized for ethanol oxidation. Formaldehyde dehydrogenase activities were investigated in Paracoccus denitrificans, Xanthobacter H4-14, and Pseudomonas AM1. P. denitrificans contained a previously reported NAD+-linked, GSH-dependent activity, but both Xanthobacter H4-14 and Pseudomonas AM1 contained numerous activities detected by activity stains of polyacrylamide gels. Induction studies showed that in Xanthobacter H4-14, a 10 kDal polypeptide, probably a dehydrogenase-associated cytochrome c, was co-induced with methanol dehydrogenase, but the formaldehyde and formate dehydrogenases were not co-regulated. Analogous induction experiments revealed similar patterns in P. denitrificans, but no evidence for co-regulation of dissimilatory activities in Pseudomonas AM1.     

甲醛氧化途径关键酶重组蛋白的生化特性及固定化酶吸收甲醛效果研究

甲醛脱氢酶(formaldehyde dehydrogenase,ADH)与甲酸脱氢酶(formate dehydrogenase,FDH)是甲醛氧化途径的两个关键酶.恶臭假单胞菌(Pseudomonas putida)的PADH是一种不依赖谷胱甘肽可以把游离甲醛直接氧化为甲酸的脱氢酶,博伊丁假丝酵母菌(Candida boidinii)的FDH在有NAD+存在时可以把甲酸氧化为二氧化碳.以基因组DNA为模板用PCR方法,从P.putida中扩增出PADH基因的编码区(padh),从C.boidinii中扩增出FDH的编码区(fdh),然后亚克隆到pET-28a(+)中分别构建这两个基因的原核表达载体pET-28a-padh和pET-28a-fdh,转化大肠杆菌,利用IPTG诱导重组蛋白PADH和FDH的表达.通过优化条件使重组蛋白的表达量占菌体总蛋白的70%以上,通过亲和层析法纯化出可溶性PADH和FDH重组蛋白.对重组蛋白的生化特性分析结果表明:PADH在最适反应温度50℃的活性为1.95 U/mg;FDH在最适反应温度40℃的活性为0.376 U/mg.所表达的重组蛋白与之前报道过的相比,具有更好的热稳定性和更广的温度适应范围.将PADH、FDH两个重组蛋白及辅因子NAD+固定到聚丙烯酰胺载体基质上,对固定化酶甲醛吸收效果的初步分析结果显示固定化酶对空气中的甲醛有一定的吸收效果,说明这两种酶被固定后具有开发成治理甲醛污染环保产品的潜力.     

Folate in demethylation: The crystal structure of the rat dimethylglycine dehydrogenase complexed with tetrahydrofolate

Dimethylglycine dehydrogenase (DMGDH) is a mammalian mitochondrial enzyme which plays an important role in the utilization of methyl groups derived from choline. DMGDH is a flavin containing enzyme which catalyzes the oxidative demethylation of dimethylglycine in vitro with the formation of sarcosine (N-methylglycine), hydrogen peroxide and formaldehyde. DMGDH binds tetrahydrofolate (THF) in vivo, which serves as an acceptor of formaldehyde and in the cell the product of the reaction is 5,10-methylenetetrahydrofolate instead of formaldehyde. To gain insight into the mechanism of the reaction we solved the crystal structures of the recombinant mature and precursor forms of rat DMGDH and DMGDH–THF complexes. Both forms of DMGDH reveal similar kinetic parameters and have the same tertiary structure fold with two domains formed by N- and C-terminal halves of the protein. The active center is located in the N-terminal domain while the THF binding site is located in the C-terminal domain about 40 Å from the isoalloxazine ring of FAD. The folate binding site is connected with the enzyme active center via an intramolecular channel. This suggests the possible transfer of the intermediate imine of dimethylglycine from the active center to the bound THF where they could react producing a 5,10-methylenetetrahydrofolate. Based on the homology of the rat and human DMGDH the structural basis for the mechanism of inactivation of the human DMGDH by naturally occurring His109Arg mutation is proposed.     

Inhibition of the low-Km mitochondrial aldehyde dehydrogenase by diethyl maleate and phorone in vivo and in vitro. Implications for formaldehyde metabolism.

Formaldehyde can be oxidized primarily by two different enzymes, the low-Km mitochondrial aldehyde dehydrogenase and the cytosolic GSH-dependent formaldehyde dehydrogenase. Experiments were carried out to evaluate the effects of diethyl maleate or phorone, agents that deplete GSH from the liver, on the oxidation of formaldehyde. The addition of diethyl maleate or phorone to intact mitochondria or to disrupted mitochondrial fractions produced inhibition of formaldehyde oxidation. The kinetics of inhibition of the low-Km mitochondrial aldehyde dehydrogenase were mixed. Mitochondria isolated from rats treated in vivo with diethyl maleate or phorone had a decreased capacity to oxidize either formaldehyde or acetaldehyde. The activity of the low-Km, but not the high-Km, mitochondrial aldehyde dehydrogenase was also inhibited. The production of CO2 plus formate from 0.2 mM-[14C]formaldehyde by isolated hepatocytes was only slightly inhibited (15-30%) by incubation with diethyl maleate or addition of cyanamide, suggesting oxidation primarily via formaldehyde dehydrogenase. However, the production of CO2 plus formate was increased 2.5-fold when the concentration of [14C]formaldehyde was raised to 1 mM. This increase in product formation at higher formaldehyde concentrations was much more sensitive to inhibition by diethyl maleate or cyanamide, suggesting an important contribution by mitochondrial aldehyde dehydrogenase. Thus diethyl maleate and phorone, besides depleting GSH, can also serve as effective inhibitors in vivo or in vitro of the low-Km mitochondrial aldehyde dehydrogenase. Inhibition of formaldehyde oxidation by these agents could be due to impairment of both enzyme systems known to be capable of oxidizing formaldehyde. It would appear that a critical amount of GSH, e.g. 90%, must be depleted before the activity of formaldehyde dehydrogenase becomes impaired.     

A novel inducible formaldehyde dehydrogenase ofPseudomonas sp. (RJ1)

A novel, pyridine-nucleotide-inducible formaldehyde dehydrogenase activity was detected in cells ofPseudomonas sp. (RJ) propagated on methylamine and oxalate. The pH optimum of the dehydrogenase was 7.0. Dichlorophenol-indophenol or potassium ferricyanide served as an electron acceptor. The rate of reduction of these electron acceptors was shown to be stimulated by phenazine methosulfate. The dehydrogenase was inhibited by parahydroxymercuric benzoate and iodoacetamide. This inhibition suggests that the enzyme contains sulfhydryl groups. The stoichiometry of the reaction in terms of oxygen uptake to formate formation was 0.5, which agrees with the theoretical value.     

Environmental regulation of alcohol metabolism in thermotolerant methylotrophic Bacillus strains

The thermotolerant methylotroph Bacillus sp. C1 possesses a novel NAD-dependent methanol dehydrogenase (MDH), with distinct structural and mechanistic properties. During growth on methanol and ethanol, MDH was responsible for the oxidation of both these substrates. MDH activity in cells grown on methanol or glucose was inversely related to the growth rate. Highest activity levels were observed in cells grown on the C₁-substrates methanol and formaldehyde. The affinity of MDH for alcohol substrates and NAD, as well as V _max, are strongly increased in the presence of a M _r 50,000 activator protein plus Mg²⁺-ions [Arfman et al. (1991) J Biol Chem 266: 3955–3960]. Under all growth conditions tested the cells contained an approximately 18-fold molar excess of (decameric) MDH over (dimeric) activator protein. Expression of hexulose-6-phosphate synthase (HPS), the key enzyme of the RuMP cycle, was probably induced by the substrate formaldehyde. Cells with high MDH and low HPS activity levels immediately accumulated (toxic) formaldehyde when exposed to a transient increase in methanol concentration. Similarly, cells with high MDH and low CoA-linked NAD-dependent acetaldehyde dehydrogenase activity levels produced acetaldehyde when subjected to a rise in ethanol concentration. Problems frequently observed in establishing cultures of methylotrophic bacilli on methanol- or ethanol-containing media are (in part) assigned to these phenomena.Abbreviations MDH NAD-dependent methanol dehydrogenase - ADH NAD-dependent alcohol dehydrogenase - A1DH CoA-linked NAD-dependent aldehyde dehydrogenase - HPS hexulose-6-phosphate synthase - G6Pdh glucose-6-phosphate dehydrogenase     

Pyruvate dehydrogenase kinases (PDKs): an overview toward clinical applications

Pyruvate dehydrogenase kinase (PDK) can regulate the catalytic activity of pyruvate decarboxylation oxidation via the mitochondrial pyruvate dehydrogenase complex, and it further links glycolysis with the tricarboxylic acid cycle and ATP generation. This review seeks to elucidate the regulation of PDK activity in different species, mainly mammals, and the role of PDK inhibitors in preventing increased blood glucose, reducing injury caused by myocardial ischemia, and inducing apoptosis of tumor cells. Regulations of PDKs expression or activity represent a very promising approach for treatment of metabolic diseases including diabetes, heart failure, and cancer. The future research and development could be more focused on the biochemical understanding of the diseases, which would help understand the cellular energy metabolism and its regulation by pharmacological effectors of PDKs.     

The role of a formaldehyde dehydrogenase-glutathione pathway in protein S-nitrosation in mammalian cells.

Intracellular sulfhydryls, both protein and non-protein, are potential targets of nitric oxide-related species. S-Nitrosation of proteins can occur in vivo and can affect their activity. Metabolic pathways that regulate protein S-nitrosation are therefore likely to be biologically important. We now report that formaldehyde dehydrogenase, an enzyme that decomposes S-nitrosoglutathione, can indirectly regulate the level of cellular protein S-nitrosation. Nitrogen oxide donors induced high levels of protein S-nitrosation in HeLa cells and lower levels in Mutatect fibrosarcoma cells, as determined by Saville-Griess assay and Western-dot-blot analysis. Depletion of glutathione by treatment with buthionine sulfoximine markedly increased protein S-nitrosation in both cell lines. Glutathione depletion also increased cytokine-induced S-nitrosation in brain endothelial cells. Formaldehyde dehydrogenase activity was 2-fold higher in Mutatect than in HeLa cells. We downregulated formaldehyde dehydrogenase activity in Mutatect cells by stably expressing antisense RNA and short-interfering RNA. In these cells, both protein S-nitrosation and S-nitrosoglutathione levels were significantly enhanced after exposure to nitrogen oxide donors as compared to parental cells. Overall, a strong inverse correlation between total S-nitrosothiols and formaldehyde dehydrogenase activity was seen. Inhibition of glutathione reductase, the enzyme that converts oxidized to reduced glutathione, by dehydroepiandrosterone similarly increased protein S-nitrosation and S-nitrosoglutathione levels in both cell lines. Our results provide the first evidence that formaldehyde dehydrogenase-dependent decomposition of S-nitrosoglutathione plays a role in protecting against nitrogen oxide-mediated protein S-nitrosation. We propose that formaldehyde dehydrogenase and glutathione reductase participate in a glutathione-dependent metabolic cycle that decreases protein S-nitrosation following exposure of cells to nitric oxide.     

Maize glutathione-dependent formaldehyde dehydrogenase cDNA: a novel plant gene of detoxification

We have previously shown that intact plants and cultured plant cells can metabolize and detoxify formaldehyde through the action of a glutathione-dependent formaldehyde dehydrogenase (FDH), followed by C-1 metabolism of the initial metabolite (formic acid). The cloning and heterologous expression of a cDNA for the glutathione-dependent formaldehyde dehydrogenase from Zea mays L. is now described. The functional expression of the maize cDNA in Escherichia coli proved that the cloned enzyme catalyses the NAD⁺- and glutathione (GSH)-dependent oxidation of formaldehyde. The deduced amino acid sequence of 41 kDa was on average 65% identical with class III alcohol dehydrogenases from animals and less than 60% identical with conventional plant alcohol dehydrogenases (ADH) utilizing ethanol. Genomic analysis suggested the existence of a single gene for this cDNA. Phylogenetic analysis supports the convergent evolution of ethanol-consuming ADHs in animals and plants from formaldehyde-detoxifying ancestors. The high structural conservation of present-day glutathione-dependent FDH in microorganisms, plants and animals is consistent with a universal importance of these detoxifying enzymes.     

Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme

The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent K _m values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde-and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPI hexulose-6-phosphate isomerase - MDH methanol dehydrogenase - ADH acohol dehydrogenase - PQQ pyrroloquinoline, quinone - DTT dithiothreitol - NBT nitrobluetetrazolium - PMS phenazine methosulphate - DCPIP dichlorophenol indophenol     

Functional coupling between vanillate-O-demethylase and formaldehyde detoxification pathway

Pseudomonas putida vanillate-O-demethylase consisting of VanA and VanB was expressed in Escherichia coli strain K-12. Recombinant E. coli strain K-12 cells expressing VanAB efficiently converted vanillate into protocatechuate with glucose consumption. Mutant lacking either pgi or zwf showed higher or lower converting activity than the parental strain, respectively. Formaldehyde, which is the by-product of the demethylation, was converted into formate in the cellular reaction. Formate accumulation was blocked by gene disruption of the E. coli frmA that coded glutathione-dependent formaldehyde dehydrogenase.