Polyethylene Glycol Dehydrogenase Research Articles

Catalytic Mechanism of Short Ethoxy Chain Nonylphenol Dehydrogenase Belonging to a Polyethylene Glycol Dehydrogenase Group in the GMC Oxidoreductase Family

Ethoxy (EO) chain nonylphenol dehydrogenase (NPEO-DH) from Ensifer sp. AS08 and EO chain octylphenol dehydrogenase from Pseudomonas putida share common molecular characteristics with polyethylene glycol (PEG) dehydrogenases (PEG-DH) and comprise a PEG-DH subgroup in the family of glucose-methanol-choline (GMC) oxidoreductases that includes glucose/alcohol oxidase and glucose/choline dehydrogenase. Three-dimensional (3D) molecular modeling suggested that differences in the size, secondary structure and hydropathy in the active site caused differences in their substrate specificities toward EO chain alkylphenols and free PEGs. Based on 3D molecular modeling, site-directed mutagenesis was utilized to introduce mutations into potential catalytic residues of NPEO-DH. From steady state and rapid kinetic characterization of wild type and mutant NPEO-DHs, we can conclude that His465 and Asn507 are directly involved in the catalysis. Asn507 mediates the transfer of proton from a substrate to FAD and His465 transfers the same proton from the reduced flavin to an electron acceptor.

Polyethylene glycol (PEG)–carboxylate–CoA synthetase is involved in PEG metabolism in Sphingopyxis macrogoltabida strain 103

Sphingopyxis macrogoltabida strain 103 possesses polyethylene-glycol (PEG)-inducible pegBCDAE operon encoding the genes relevant to PEG degradation. PEG is converted to PEG-carboxylate by PegA (PEG dehydrogenase) and PegC (PEG-aldehyde dehydrogenase). In this study, the recombinant PegE (homologous to acyl-CoA synthetases) was characterized. PegE was an acyl-CoA synthetase active for PEG-carboxylate and fatty acids. Judging from the nature of this kind of protein (located on the cytoplasmic membrane as a translocator), PegE might be responsible for the translocation of PEG-carboxylate from the periplasm into the cytoplasm or for the detoxification of strong acidity of the substrate.

Isolation of bacteria able to grow on both polyethylene glycol (PEG) and polypropylene glycol (PPG) and their PEG/PPG dehydrogenases

Two bacterial consortia growing on a random copolymer of ethylene glycol and propylene glycol units were obtained by enrichment cultures from various microbial samples. Six major strains included in both consortia were purified and identified as Sphingomonads, Pseudomonas sp. and Stenotrophomonas maltophilia. Three of them (Sphingobium sp. strain EK-1, Sphingopyxis macrogoltabida strain EY-1, and Pseudomonas sp. strain PE-2) utilized both PEG and polypropylene glycol (PPG) as a sole carbon source. Four PEG-utilizing bacteria had PEG dehydrogenase (PEG-DH) activity, which was induced by PEG. PCR products from DNA of these bacteria generated with primers designed from a PEG-DH gene (AB196775 for S. macrogoltabida strain 103) indicated the presence of a sequence that is the homologous to the PEG-DH gene (99% identity). On the other hand, five PPG-utilizing bacteria had PPG dehydrogenase (PPG-DH) activity, but the activity was constitutive. PCR of a PPG-DH gene was performed using primers designed from a polyvinyl alcohol dehydrogenase (PVA-DH) gene (AB190288 for Sphingomonas sp. strain 113P3) because a PPG-DH gene has not been cloned yet, but both PPG-DH and PVA-DH were active toward PPG and PVA (Mamoto et al. 2006). PCR products of the five strains did not have similarity to each other or to oxidoreductases including PVA-DH.

Analysis of Amino Acid Residues Involved in Catalysis of Polyethylene Glycol Dehydrogenase from Sphingopyxis terrae , Using Three-Dimensional Molecular Modeling-Based Kinetic Characterization of Mutants

Polyethylene glycol dehydrogenase (PEGDH) from Sphingopyxis terrae (formerly Sphingomonas terrae) is composed of 535 amino acid residues and one flavin adenine dinucleotide per monomer protein in a homodimeric structure. Its amino acid sequence shows 28.5 to 30.5% identity with glucose oxidases from Aspergillus niger and Penicillium amagasakiense. The ADP-binding site and the signature 1 and 2 consensus sequences of glucose-methanol-choline oxidoreductases are present in PEGDH. Based on three-dimensional molecular modeling and kinetic characterization of wild-type PEGDH and mutant PEGDHs constructed by site-directed mutagenesis, residues potentially involved in catalysis and substrate binding were found in the vicinity of the flavin ring. The catalytically important active sites were assigned to His-467 and Asn-511. One disulfide bridge between Cys-379 and Cys-382 existed in PEGDH and seemed to play roles in both substrate binding and electron mediation. The Cys-297 mutant showed decreased activity, suggesting the residue's importance in both substrate binding and electron mediation, as well as Cys-379 and Cys-382. PEGDH also contains a motif of a ubiquinone-binding site, and coenzyme Q10 was utilized as an electron acceptor. Thus, we propose several important amino acid residues involved in the electron transfer pathway from the substrate to ubiquinone.

A new ether bond-splitting enzyme found in Gram-positive polyethylene glycol 6000-utilizing bacterium, Pseudonocardia sp. strain K1

Pseudonocardia sp. strain K1 is the only Gram-positive bacterium among the bacteria aerobically metabolizing polyethylene glycol (PEG). Generally, PEG is metabolized by an oxidative pathway in which a terminal alcohol group of PEG is oxidized to aldehyde and to carboxylic acid and then an ether bond is oxidatively cleaved. As the cell-free extract of Pseudonocardia sp. strain K1 has PEG dehydrogenase, PEG aldehyde dehydrogenase and diglycolic acid (DGA) dehydrogenase (DGADH) activities, all of which are constitutively formed, the strain has a metabolic pathway similar to that so far known. We purified an ether bond-splitting enzyme as DGADH. The molecular mass of the enzyme was estimated to be 55 kDa; and it consisted of two identical subunits. The enzyme oxidatively cleaved both an ether bond of PEG 3000 dicarboxylic acid and DGA. The N-terminal amino acid sequence of the purified enzyme had high homology with various superoxide dismutases and the enzyme had also superoxide dismutase activity. The atomic absorption spectrum showed that approximately one atom of Fe was included in each subunit of the enzyme. DGADH activity increased in the cells grown in a PEG medium supplemented with FeCl(3). Thus, we concluded that the enzyme purified from Pseudonocardia sp. strain K1 is a new ether bond-splitting enzyme.

Microbial degradation of polyethers.

This paper summarizes studies on microbial degradation of polyethers. Polyethers are aerobically metabolized through common mechanisms (oxidation of terminal alcohol groups followed by terminal ether cleavage), well-characterized examples being found with polyethylene glycol (PEG). First the polymer is oxidized to carboxylated PEG by alcohol and aldehyde dehydrogenases and then the terminal ether bond is cleaved to yield the depolymerized PEG by one glycol unit. Most probably PEG is anaerobically metabolized through one step which is catalyzed by PEG acetaldehyde lyase, analogous to diol dehydratase. Whether aerobically or anaerobically, the free OH group is necessary for metabolization of PEG. PEG with a molecular weight of up to 20,000 was metabolized either in the periplasmic space (Pseudomonas stutzeri and sphingomonads) or in the cytoplasm (anaerobic bacteria), which suggests the transport of large PEG through the outer and inner membranes of Gram-negative bacterial cells. Membrane-bound PEG dehydrogenase (PEG-DH) with high activity towards PEG 6,000 and 20,000 was purified from PEG-utilizing sphingomonads. Sequencing of PEG-DH revealed that the enzyme belongs to the group of GMC flavoproteins, FAD being the cofactor for the enzyme. On the other hand, alcohol dehydrogenases purified from other bacteria that cannot grow on PEG oxidized PEG. Cytoplasmic NAD-dependent alcohol dehydrogenases with high specificity towards ether-alcohol compound, either crude or purified, showed appreciable activity towards PEG 400 or 600. Liver alcohol dehydrogenase (equine) also oxidized PEG homologs, which might cause fatal toxic syndrome in vivo by carboxylating PEG together with aldehyde dehydrogenase when PEG was absorbed. An ether bond-cleaving enzyme was detected in PEG-utilizing bacteria and purified as diglycolic acid (DGA) dehydrogenase from a PEG-utilizing consortium. The enzyme oxidized glycolic acid, glyoxylic acid, as well as PEG-carboxylic acid and DGA. Similarly, dehydrogenation on polypropylene glycol (PPG) and polytetramethylene glycol (PTMG) was suggested with cell-free extracts of PPG and PTMG-utilizing bacteria, respectively. PPG commercially available is atactic and includes many structural (primary and secondary alcohol groups) and optical (derived from pendant methyl groups on the carbon backbone) isomers. Whether PPG dehydrogenase (PPG-DH) has wide stereo- and enantioselective substrate specificity towards PPG isomers or not must await further purification. Preliminary research on PPG-DH revealed that the enzyme was inducibly formed by PPG in the periplasmic, membrane and cytoplasm fractions of a PPG-utilizing bacterium Stenotrophomonas maltophilia. This finding indicated the intracellular metabolism of PPG is the same as that of PEG. Besides metabolization of polyethers, a biological Fenton mechanism was proposed for degradation of PEG, which was caused by extracellular oxidants produced by a brown-rot fungus in the presence of a reductant and Fe3+, although the metabolism of fragmented PEG has not yet been well elucidated.

Role of novel dye-linked dehydrogenases in the metabolism of polyethylene glycol by pure cultures of Sphingomonas sp. N6

Polyethylene glycol and diglycolic acid dehydrogenase acitivities linked with3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2 H-tetrazolium bromide and phenazine methosulfate were found in the participate fraction of the sonic extract of a newly isolated polyethylene glycol 20 000-utilizing bacterium ( Sphingomonas sp. N6). The amount of glyoxylic acid formed from diglycolic acid increased proportionally with the increase in reaction time and enzyme concentration to show that diglycolic acid can be a model compound for an ether-cleaving enzyme. Both enzymes were formed inducibly when the organism was grown on polyethylene glycol 10000. Both enzymes transferred many more electrons to3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2 H-tetrazolium bromide plus phenazine methosulfate than to 2,6-dichlorophenolindophenol plus phenazine methosulfate. Also, ferricyanide, nitroblue tetrazolium and horse heart cytochrome c served as electron acceptors, among which3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide plus phenazine methosulfate was most active for polyethylene glycol dehydrogenase and ferricyanide was most active for diglycolic acid dehydrogenase irrespective of the presence of phenazine methosulfate. NAD and NADP did not act as electron acceptors. Polyethylene glycol dehydrogenase was completely inhibited by 1,4-benzoquinone and partially inhibited by quinine and glyoxylic acid. Diglycolic acid dehydrogenase was strongly inhibited by 1,4-benzoquinone and partially inhibited by α-benzoin oxime, quinacrine and glyoxylic acid. The enzymes appear to be different from each other and also from polyethylene glycol and diglycolic acid dehydrogenases of polyethylene glycol 20 000-utilizing symbiotic mixed culture E-1in which S. terrae was responsible for polyethylene glycol degradation in the coupling with electron acceptors and in the effect of inhibitors.

Role of novel dye-linked dehydrogenases in the metabolism of polyethylene glycol by pure cultures ofSphingomonassp. N6

Abstract Polyethylene glycol and diglycolic acid dehydrogenase acitivities linked with3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2 H-tetrazolium bromide and phenazine methosulfate were found in the participate fraction of the sonic extract of a newly isolated polyethylene glycol 20 000-utilizing bacterium ( Sphingomonas sp. N6). The amount of glyoxylic acid formed from diglycolic acid increased proportionally with the increase in reaction time and enzyme concentration to show that diglycolic acid can be a model compound for an ether-cleaving enzyme. Both enzymes were formed inducibly when the organism was grown on polyethylene glycol 10000. Both enzymes transferred many more electrons to3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2 H-tetrazolium bromide plus phenazine methosulfate than to 2,6-dichlorophenolindophenol plus phenazine methosulfate. Also, ferricyanide, nitroblue tetrazolium and horse heart cytochrome c served as electron acceptors, among which3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide plus phenazine methosulfate was most active for polyethylene glycol dehydrogenase and ferricyanide was most active for diglycolic acid dehydrogenase irrespective of the presence of phenazine methosulfate. NAD and NADP did not act as electron acceptors. Polyethylene glycol dehydrogenase was completely inhibited by 1,4-benzoquinone and partially inhibited by quinine and glyoxylic acid. Diglycolic acid dehydrogenase was strongly inhibited by 1,4-benzoquinone and partially inhibited by α-benzoin oxime, quinacrine and glyoxylic acid. The enzymes appear to be different from each other and also from polyethylene glycol and diglycolic acid dehydrogenases of polyethylene glycol 20 000-utilizing symbiotic mixed culture E-1in which S. terrae was responsible for polyethylene glycol degradation in the coupling with electron acceptors and in the effect of inhibitors.

Polyethylene glycol dehydrogenase activity of Rhodopseudomonas acidophila derives from a type I quinohaemoprotein alcohol dehydrogenase

Dye-linked alcohol dehydrogenase from Rhodopseudomonas acidophila strain M402, able to oxidize polyethylene glycols, was purified to homogeneity. The monomeric enzyme, having a molecular mass of 72 kDa, contains one PQQ and one haem c per enzyme molecule. In other respects also, the enzyme is very similar to the type I quinohaemoprotein alcohol dehydrogenases known to occur in Comamonas testosteroni, Comamonas acidovorans, and Pseudomonas putida species. However, dissimilarities exist with respect to the isoelectric points and the substrate specificities. On reinvestigating the substrate specificity of the C. testosteroni enzyme, it also appeared to exhibit good activity towards polyethylene glycols. Based on what has been reported for the polyethylene glycol-oxidizing alcohol dehydrogenase of Sphingomonas macrogoltabidus, this enzyme is quite different from that of R. acidophila. Keywords: Polyethylene glycol dehydrogenase activity; Alcohol dehydrogenase; PQQ; Haem c; Rhodopseudomonas acidophila

Polyethylene glycol dehydrogenase activity ofRhodopseudomonas acidophiladerives from a type I quinohaemoprotein alcohol dehydrogenase

Dye-linked alcohol dehydrogenase from Rhodopseudomonas acidophila strain M402, able to oxidize polyethylene glycols, was purified to homogeneity. The monomeric enzyme, having a molecular mass of 72 kDa, contains one PQQ and one haem c per enzyme molecule. In other respects also, the enzyme is very similar to the type I quinohaemoprotein alcohol dehydrogenases known to occur in Comamonas testosteroni, Comamonas acidovorans, and Pseudomonas putida species. However, dissimilarities exist with respect to the isoelectric points and the substrate specificities. On reinvestigating the substrate specificity of the C. testosteroni enzyme, it also appeared to exhibit good activity towards polyethylene glycols. Based on what has been reported for the polyethylene glycol-oxidizing alcohol dehydrogenase of Sphingomonas macrogoltabidus, this enzyme is quite different from that of R. acidophila. Keywords: Polyethylene glycol dehydrogenase activity; Alcohol dehydrogenase; PQQ; Haem c; Rhodopseudomonas acidophila

Polyethylene glycol dehydrogenating activity demonstrated by dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila M402.

Ethylene glycols and polyethylene glycols inhibited the growth of a photosynthetic bacterium, Rhodopseudomonas acidophila, strain M402 under aerobic-dark and anaerobic-light culture conditions. However, polyethylene glycol dehydrogenating activity was demonstrated with phenazine methosulfate (PMS) as an electron acceptor in parallel with the PMS-linked aromatic alcohol dehydrogenase activity. Addition of ethylene glycols or polyethylene glycols to the culture neither induced aromatic alcohol dehydrogenase, nor accelerated polyethylene glycol dehydrogenating activity. Purified PMS-linked aromatic alcohol dehydrogenase had high affinities toward diethylene glycol and polyethylene glycols. This indicated that the dye-linked aromatic alcohol dehydrogenase is capable of oxidizing these xenobiotic synthetic polymer alcohols, polyethylene glycols. There is no evidence on the existence of a specific polyethylene glycol dehydrogenase in this bacterium.

Purification and characterization of constitutive polyethylene glycol (PEG) dehydrogenase of a PEG 4000-utilizing Flavobacterium sp. no. 203

Polyethylene glycol (PEG) 4000-utilizing bacterium no. 203 was identified as a Flavobacterium species. 2, 6-Dichlorophenol-indophenol (DCIP)-dependent PEG dehydrogenase was constitutively formed in nutrient broth, glucose and PEG media. However, the enzyme formation was repressed in the presence of an excess amount (over 0.25%) of PEG 400 or 1000. PEG dehydrogenase was purified approximately 34 fold by precipitation with ammonium sulfate, solubilization with benzalkonium chloride, chromatography with DEAE-Toyopearl 650 M and hydroxylapatite and gel filtration on Toyopearl HW-55. The molecular weight of the purified PEG dehydrogenase was calculated to be approximately 2.20 × 10 5, a value which seemed to consist of four subunits with the same molecular weight of 5.70 × 10 4. The enzyme was stable below 40°C and in the pH range of 7.0 and 8.0. The optimum pH and temperature of the activity were around 8.0 and 40°C, respectively. The enzyme reduced DCIP and coenzyme Q 1 and Q 2. PEG dehydrogenase showed activity toward various PEG molecules (dimer-PEG 20,000). The apparent K m values for PEG 400, 1000, 4000 and 6000 were about 1.0, 1.7, 2.8 and 5.9 mM, respectively. The enzyme oxidized primary aliphatic alcohols of C 3–C 12, the corresponding aldehydes of C 3–C 7, aromatic alcohols and aldehydes, diols, etc. The enzyme was inactive on ethylene glycol, glycerol, secondary alcohols and sugar alcohols. The enzyme activity was strongly inhibited by sulfhydryl agents or heavy metals and 1, 4-benzoquinone. The purified enzyme showed absorption apectrum similar to that of PEG 6000 dehydrogenase which has already been reported to be a quinoprotein. The prosthetic group of the enzyme was extracted with methanol and identified as PQQ from its prosthetic group capability for glucose dehydrogenase and the fluorescence spectrum.

Inducible or constitutive polyethylene glycol dehydrogenase involved in the aerobic metabolism of polyethylene glycol

2, 6-Dichlorophenolindophenol (DCIP)-dependent polyethylene glycol (PEG) dehydrogenase activity was found in the particulate fractions of cell-free extracts prepared from PEG-utilizing bacteria ( Pseudomonas and Flavobacterium species). This result suggested that PEG dehydrogenase is linked to the respiratory chain of each bacterium and that the enzyme plays a major role in the aerobic metabolism of PEG. Enzyme activities were strongly inhibited by 1, 4-benzoquinone. No metal ion was indispensable for the enzyme activities. Enzyme activities of PEG-utilizing bacteria were induced by PEG except for the activity of PEG 4000-utilizing Flavobacterium sp. no. 203 which had a constitutive enzyme. Although PEG-utilizing bacteria had different growth substrate specificities toward PEGs 200–20,000, their PEG dehydrogenases oxidized the same molecular wt. range of PEGs (dimer-20,000). Cell-free extracts of PEG 400-, 1000- or 4000-utilizing bacteria oxidized PEG 6000 and 20,000 though these bigger PEGs could not be utilized as the sole carbon and energy sources by the bacteria. Methanol, ethylene glycol and glycerol were not or only barely dehydrogenated by all the enzyme preparations.

Biodegradation of polyethylene glycol by symbiotic mixed culture (obligate mutualism)

Neither Flavobacterium sp. nor Pseudomonas sp. grew on a polyethylene glycol (PEG) 6000 medium containing the culture filtrate of their mixed culture on PEG 6000. The two bacteria did not grow with a dialysis culture on a PEG 6000 medium. Flavobacterium sp. grew well on a dialysis culture containing a tetraethylene glycol medium supplemented with a small amount of PEG 6000 as an inducer, while poor growth of Pseudomonas sp. was observed. Three enzymes involved in the metabolism of PEG, PEG dehydrogenase, PEG-aldehyde dehydrogenase and PEG-carboxylate dehydrogenase (ether-cleaving) were present in the cells of Flavobacterium sp. The first two enzymes were not found in the cells of Pseudomonas sp. PEG 6000 was degraded neither by intact cells of Flavobacterium sp. nor by those of Pseudomonas sp., but it was degraded by their mixture. Glyoxylate, a metabolite liberated by the ether-cleaving enzyme, inhibited the growth of the mixed culture. The ether-cleaving enzyme was remarkably inhibited by glyoxylate. Glyoxylate was metabolized faster by Pseudomonas sp. than by Flavobacterium sp., and seemed to be a key material for the symbiosis.

Identification of the prosthetic group and further characterization of a novel enzyme, polyethylene glycol dehydrogenase.

The purified polyethylene glycol (PEG) dehydrogenase from cells of a synergistic mixed culture of Flavobacterium and Pseudomonas species showed a similar absorption spectrum to those of other quinoproteins reported so far. The prosthetic group of the PEG dehydrogenase after extraction with cold methanol and purification by DEAE-Sephadex A-25 column chromatography and Sephadex G-25 gel filtration showed the same elution profiles as those of authentic pyrrolo-quinoline quinone (PQQ). Absorption and fluorescence spectra of the purified prosthetic group and its prosthetic group capability for glucose dehydrogenase indicated that it was identical with authentic PQQ.The enzyme was induced during bacterial cell growth on a medium containing PEG 6000 as a sole source of carbon. The purified enzyme oxidized primary alcohols of C2-C16 and the corresponding aldehydes of C4-C7. The enzyme also reacted with nonionic surfactants containing PEG residues. The enzyme reduced 2,6-dichlorophenolindophenol (DCIP) and the Km ...

Identification of reaction products of polyethylene glycol dehydrogenase.

Identification of reaction products of polyethylene glycol dehydrogenase.

Purification and characterization of polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene glycol.

Polyethylene glycol (PEG) dehydrogenase in crude extracts of a PEG 20,000-utilizing mixed culture was purified 24 times by precipitation with ammonium sulfate, solubilization with laurylbetaine, and chromatography with diethylamino-ethyl-cellulose, hydroxylapatite, and Sephadex G-200. The purified enzyme was confirmed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme, which appeared to consist of four identical subunits, was 2.4 X 10(5). The enzyme was stable below 35 degrees C and in the pH range of 7.5 to 9.0. The optimum pH and temperature of the activity were around 8.0 and 60 degrees C, respectively. The enzyme did not require any metal ions for activity and oxidized various kinds of PEGs, among which PEG 6,000 was the most active substrate. The apparent Km values for tetraethylene glycol and PEG 6,000 were about 10.0 and 3.0 mM, respectively.

Bacterial oxidation of polyethylene glycol.

The metabolism of polyethylene glycol (PEG) was investigated with a synergistic, mixed culture of Flavobacterium and Pseudomonas species, which are individually unable to utilize PEGs. The PEG dehydrogenase linked with 2,6-dichlorophenolindophenol was found in the particulate fraction of sonic extracts and catalyzed the formation of a 2,4-dinitrophenylhydrazine-positive compound, possibly an an aldehyde. The enzyme has a wide substrate specificity towards PEGs: from diethylene glycol to PEG 20,000 Km values for tetraethylene glycol (TEG), PEG 400, and PEG 6,000 were 11, 1.7, and 15 mM, respectively. The metabolic products formed from TEG by intact cells were isolated and identified by combined gas chromatography-mass spectrometry as triethylene glycol and TEG-monocarboxylic acid plus small amounts of TEG-dicarboxylic acid, diethylene glycol, and ethylene glycol. From these enzymatic and analytical data, the following metabolic pathway was proposed for PEG: HO(CH2CH2O)nCH2CH2OH leads to HO(CH2CH2O)nCH2CHO leads to HO(CH2CH2O)nCH2COOH leads to HO(CH2CH2O)n-1CH2CH2OH.