Quinoprotein Alcohol Dehydrogenase Research Articles

β-oxidation–polyhydroxyalkanoates synthesis relationship in Pseudomonas putida KT2440 revisited

Pseudomonas putida KT2440 is a well-known model organism for the medium-chain-length (mcl) polyhydroxyalkanoate (PHA) accumulation. (R)-Specific enoyl-coenzyme A hydratase (PhaJ) was considered to be the main supplier of monomers for PHA synthesis by converting the β-oxidation intermediate, trans-2-enoyl-CoA to (R)-3-hydroxyacyl-CoA when fatty acids (FA) are used. Three PhaJ homologues, PhaJ1, PhaJ4 and MaoC, are annotated in P. putida KT2440. To investigate the relationship of fatty acids–PHA metabolism and the role of each PhaJ in PHA biosynthesis in P. putida KT2440, a series of P. putida KT2440 knockouts was obtained. PHA content and monomer composition in wild type (WT) and mutants under different growth conditions were analysed. PhaJ4 was the main monomer supplier for PHA synthesis with FA as sole carbon and energy source, with preference towards C8 and C10 substrate, whereas PhaJ1 showed preference for the C6 substrate. However, when all three PhaJ homologues were deleted, the mutant still accumulated PHA up to 10.7% of the cell dry weight (CDW). The deletion of (R)-3-hydroxydecanoyl-ACP:CoA transacylase (PhaG), which connects de novo FA and PHA synthesis pathways, while causing a further 1.8-fold decrease in PHA content, did not abolish PHA accumulation. Further proteome analysis revealed quinoprotein alcohol dehydrogenases PedE and PedH as potential monomer suppliers, but when these were deleted, the PHA level remained at 2.2–14.8% CDW depending on the fatty acid used and whether nitrogen limitation was applied. Therefore, it is likely that some other non-specific dehydrogenases supply monomers for PHA synthesis, demonstrating the redundancy of PHA metabolism.Key points• β-oxidation intermediates are converted to PHA monomers by hydratases PhaJ1, PhaJ4 and MaoC in Pseudomonas putida KT2440.• When these are deleted, the PHA level decreases, but it is not abolished.• PHA non-specific enzyme(s) also contributes to PHA metabolism in KT2440.

Dehydrogenation Mechanism of Three Stereoisomers of Butane-2,3-Diol in Pseudomonas putida KT2440.

Pseudomonas putida KT2440 is a promising chassis of industrial biotechnology due to its metabolic versatility. Butane-2,3-diol (2,3-BDO) is a precursor of numerous value-added chemicals. It is also a microbial metabolite which widely exists in various habiting environments of P. putida KT2440. It was reported that P. putida KT2440 is able to use 2,3-BDO as a sole carbon source for growth. There are three stereoisomeric forms of 2,3-BDO: (2R,3R)-2,3-BDO, meso-2,3-BDO and (2S,3S)-2,3-BDO. However, whether P. putida KT2440 can utilize three stereoisomeric forms of 2,3-BDO has not been elucidated. Here, we revealed the genomic and enzymic basis of P. putida KT2440 for dehydrogenation of different stereoisomers of 2,3-BDO into acetoin, which will be channeled to central mechanism via acetoin dehydrogenase enzyme system. (2R,3R)-2,3-BDO dehydrogenase (PP0552) was detailedly characterized and identified to participate in (2R,3R)-2,3-BDO and meso-2,3-BDO dehydrogenation. Two quinoprotein alcohol dehydrogenases, PedE (PP2674) and PedH (PP2679), were confirmed to be responsible for (2S,3S)-2,3-BDO dehydrogenation. The function redundancy and inverse regulation of PedH and PedE by lanthanide availability provides a mechanism for the adaption of P. putida KT2440 to variable environmental conditions. Elucidation of the mechanism of 2,3-BDO catabolism in P. putida KT2440 would provide new insights for bioproduction of 2,3-BDO-derived chemicals based on this robust chassis.

Phenanthrene degradation by the bacterium Pseudomonas stutzeri JP1 under low oxygen condition

Phenanthrene, is one of the important polycyclic aromatic hydrocarbons (PAHs), which can be used as indicator for measuring PAHs and as a model molecule for studying PAHs biodegradation. During phenanthrene bioremediation bacterial species plays an important. In present work, a previously isolated PAHs-degrading bacterium, Pseudomonas stutzeri JP1, was used to investigate the phenanthrene degradation mechanism under low oxygen condition. RNA-sequencing was conducted for gene detections. The results indicated that methylcrotonoyl-CoA carboxylase gene and quinoprotein alcohol dehydrogenase genes of bacterium were up-regulated during the PAHs exposure. The results of RT-qPCR revealed that the genes encoding the quinoprotein alcohol dehydrogenase were 3.16-fold up-regulated. Further, two-dimensional electrophoresis (2-DE) and MALDI-TOF-TOF were used to study the proteins related to phenanthrene degradation. The results showed that expression of six proteins obviously increased upon phenanthrene induction. Quinoprotein alcohol dehydrogenase gene was involved in phenanthrene catabolism. The results correspond nicely to the RNA-Seq data. This work provided in-depth knowledge as well as technical support for bioremediation of polycyclic aromatic hydrocarbons contamination via microbes in low oxygen environment.

Regulation of a Glycerol-Induced Quinoprotein Alcohol Dehydrogenase by σ54 and a LuxR-Type Regulator in Azospirillum brasilense Sp7.

Azospirillum brasilense Sp7 uses glycerol as a carbon source for growth and nitrogen fixation. When grown in medium containing glycerol as a source of carbon, it upregulates the expression of a protein which was identified as quinoprotein alcohol dehydrogenase (ExaA). Inactivation of exaA adversely affects the growth of A. brasilense on glycerol. A determination of the transcription start site of exaA revealed an RpoN-dependent -12/-24 promoter consensus. The expression of an exaA::lacZ fusion was induced maximally by glycerol and was dependent on σ54 Bioinformatic analysis of the sequence flanking the -12/-24 promoter revealed a 17-bp sequence motif with a dyad symmetry of 6 nucleotides upstream of the promoter, the disruption of which caused a drastic reduction in promoter activity. The electrophoretic mobility of a DNA fragment containing the 17-bp sequence motif was retarded by purified EraR, a LuxR-type transcription regulator that is transcribed divergently from exaA EraR also showed a positive interaction with RpoN in two-hybrid and pulldown assays.IMPORTANCE Quinoprotein alcohol dehydrogenase (ExaA) plays an important role in the catabolism of alcohols in bacteria. Although exaA expression is thought to be regulated by a two-component system consisting of EraS and EraR, the mechanism of regulation was not known. This study shows the details of the regulation of expression of the exaA gene in A. brasilense We have shown here that exaA of A. brasilense is maximally induced by glycerol and harbors a σ54-dependent promoter. The response regulator EraR binds to an inverted repeat located upstream of the exaA promoter. This study shows that a LuxR-type response regulator (EraR) binds upstream of the exaA gene and physically interacts with σ54 The unique feature of this regulation is that EraR is a LuxR-type transcription regulator that lacks the GAFTGA motif, a characteristic feature of the enhancer binding proteins that are known to interact with σ54 in other bacteria.

Analysis of the molecular response of Pseudomonas putida KT2440 to the next-generation biofuel n-butanol

To increase the efficiency of biocatalysts a thorough understanding of the molecular response of the biocatalyst to precursors, products and environmental conditions applied in bioconversions is essential. Here we performed a comprehensive proteome and phospholipid analysis to characterize the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the next-generation biofuel n-butanol. Using complementary quantitative proteomics approaches we were able to identify and quantify 1467 proteins, corresponding to 28% of the total KT2440 proteome. 256 proteins were altered in abundance in response to n-butanol. The proteome response entailed an increased abundance of enzymes involved in n-butanol degradation including quinoprotein alcohol dehydrogenases, aldehyde dehydrogenases and enzymes of fatty acid beta oxidation. From these results we were able to construct a pathway for the metabolism of n-butanol in P. putida. The initial oxidation of n-butanol is catalyzed by at least two quinoprotein ethanol dehydrogenases (PedE and PedH). Growth of mutants lacking PedE and PedH on n-butanol was significantly impaired, but not completely inhibited, suggesting that additional alcohol dehydrogenases can at least partially complement their function in KT2440. Furthermore, phospholipid profiling revealed a significantly increased abundance of lyso-phospholipids in response to n-butanol, indicating a rearrangement of the lipid bilayer. Biological significancen-butanol is an important bulk chemical and a promising alternative to gasoline as a transportation fuel. Due to environmental concerns as well as increasing energy prices there is a growing interest in sustainable and cost-effective biotechnological production processes for the production of bulk chemicals and transportation fuels from renewable resources. n-butanol fermentation is well established in Clostridiae, but the efficiency of n-butanol production is mainly limited by its toxicity. Therefore bacterial strains with higher intrinsic tolerance to n-butanol have to be selected as hosts for n-butanol production. Pseudomonas bacteria are metabolically very versatile and exhibit a high intrinsic tolerance to organic solvents making them suitable candidates for bioconversion processes. A prerequisite for a potential production of n-butanol in Pseudomonas bacteria is a thorough understanding of the molecular adaption processes caused by n-butanol and the identification of enzymes involved in n-butanol metabolization. This work describes the impact of n-butanol on the proteome and the phospholipid composition of the reference strain P. putida KT2440. The high proteome coverage of our proteomics survey allowed us to reconstruct the degradation pathway of n-butanol and to monitor the changes in the energy metabolism of KT2440 induced by n-butanol. Key enzymes involved in n-butanol degradation identified in study will be interesting targets for optimization of n-butanol production in Pseudomonads. The present work and the identification of key enzymes involved in butanol metabolism may serve as a fundament to develop new or improve existing strategies for the biotechnological production of the next-generation biofuel n-butanol in Pseudomonads.

Effect of amines as activators on the alcohol-oxidizing activity of pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenase.

Pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenases (PQQ-ADH) require ammonia or primary amines as activators in in vitro assays with artificial electron acceptors. We found that PQQ-ADH from Pseudomonas putida KT2440 (PpADH) was activated by various primary amines, di-methylamine, and tri-methylamine. The alcohol oxidation activity of PpADH was strongly enhanced and the affinity for substrates was also improved by pentylamine as an activator.

Isolation and characterization of Pseudomonas sp. strain capable of degrading diethylstilbestrol

Since diethylstilbestrol (DES) interrupts endocrine systems and generates reproductive abnormalities in both wildlife and human beings, methods to remove DES from the environments are urgently recommended. In this study, bacterial strain J51 was isolated and tested to effectively degrade DES. J51 was identified as Pseudomonas sp. based on its nucleotide sequence of 16S rRNA. The quinoprotein alcohol dehydrogenase and isocitrate lyase were identified to be involved in DES degradation by MALDI-TOF-TOF MS/MS analysis. In the presence of 40 mg/l DES, increase of the genes encoding quinoprotein alcohol dehydrogenase and isocitrate lyase in both RNA and protein levels was determined. The HPLC/MS analysis showed that DES was hydrolyzed to a major degrading metabolite DES-4-semiquinone. It was the first time to demonstrate the characteristics of DES degradation by specific bacterial strain and the higher degradation efficiency indicated the potential application of Pseudomonas sp. strain J51 in the treatment of DES-contaminated freshwater and seawater environments.

Cloning and functional analysis of adhS gene encoding quinoprotein alcohol dehydrogenase subunit III from Acetobacter pasteurianus SKU1108

The adhS gene which encodes the smallest subunit, subunit III, of quinoprotein alcohol dehydrogenase (PQQ-ADH) from Acetobacter pasteurianus SKU1108 has been cloned and characterized. The role of this subunit on the function of PQQ-ADH was investigated by construction of adhS gene disruptant and mutants. The adhS gene disruptant completely lost its PQQ-ADH activity and acetate-producing ability but retained acetic acid toleration. In contrast, this disruptant grew well, even better than the wild type, in the ethanol containing medium even though its PQQ-ADH activity and ethanol oxidizing ability was completely lost, while NAD +-dependent ADH (NAD +-ADH) was induced. Heme staining and immunoblot analysis of both membrane and soluble fractions with anti-ADH subunit III suggested that ethanol did not affect the adhS gene expression but induced PQQ-ADH activity. Over-expressed adhS did not enhance acetic acid production in both the wild type and the adhS disruptant. In addition, deletion analysis of upstream region of adhS gene suggested that its tentative promoter(s) might be located at around 118–268 bp upstream from an initiation codon. Random mutagenesis of adhS gene revealed that complete loss of PQQ-ADH activity and ethanol oxidizing ability were observed in the mutants' lack of the 140 and 73 amino acid residues at the C-terminal, whereas the lack of 22 amino acid residues at the C-terminal affected neither the PQQ-ADH activity nor ethanol oxidizing ability. In addition, some amino acid substitutions such as Leu18Gln, Ala26Val, Val36Ile, Val54Ile, Gly55Asp, Val70Ala and Val107Ala did not show any affect on PQQ-ADH activity and ethanol oxidizing ability. Interestingly, alteration of Thr104Lys led to a complete loss of ethanol oxidizing ability. However, point mutation at the possible promoter region also exhibited low PQQ-ADH activity and ethanol oxidizing ability. This result suggests that 104Thr might be involved in molecular coupling with subunit I in order to construct active ADH complex, whereas 22 amino acid residues at C-terminal may be not necessary for PQQ-ADH activity.

Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases

We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41 degrees C, tolerate to 1.5% acetic acid or 4% ethanol at 39 degrees C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37 degrees C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4-5% ethanol at the same temperature. The fermentation ability at 37 degrees C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37 degrees C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.

Analysis of the promoter activities of the genes encoding three quinoprotein alcohol dehydrogenases in Pseudomonas putida HK5

The transcriptional regulation of three distinct alcohol oxidation systems, alcohol dehydrogenase (ADH)-I, ADH-IIB and ADH-IIG, in Pseudomonas putida HK5 was investigated under various induction conditions. The promoter activities of the genes involved in alcohol oxidation were determined using a transcriptional lacZ fusion promoter-probe vector. Ethanol was the best inducer for the divergent promoters of qedA and qedC, encoding ADH-I and a cytochrome c, respectively. Primary and secondary C3 and C4 alcohols and butyraldehyde specifically induced the divergent promoters of qbdBA and aldA, encoding ADH-IIB and an NAD-dependent aldehyde dehydrogenase, respectively. The qgdA promoter of ADH-IIG responded well to (S)-(+)-1,2-propanediol induction. In addition, the roles of genes encoding the response regulators exaE and agmR, located downstream of qedA, were inferred from the properties of exaE- or agmR-disrupted mutants and gene complementation tests. The gene products of both exaE and agmR were strictly necessary for qedA transcription. The mutation and complementation studies also suggested a role for AgmR, but not ExaE, in the transcriptional regulation of qbdBA (ADH-IIB) and qgdA (AGH-IIG). A hypothetical scheme describing a regulatory network, which directs expression of the three distinct alcohol oxidation systems in P. putida HK5, was derived.

A Tightly Bound Quinone Functions in the Ubiquinone Reaction Sites of Quinoprotein Alcohol Dehydrogenase of an Acetic Acid Bacterium,Gluconobacter suboxydans

Quinoprotein alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound enzyme that functions as the primary dehydrogenase in the ethanol oxidase respiratory chain. It consists of three subunits and has a pyrroloquinoline quinone (PQQ) in the active site and four heme c moieties as electron transfer mediators. Of these, three heme c sites and a further site have been found to be involved in ubiquinone (Q) reduction and ubiquinol (QH2) oxidation respectively (Matsushita et al., Biochim. Biophys. Acta, 1409, 154-164 (1999)). In this study, it was found that ADH solubilized and purified with dodecyl maltoside, but not with Triton X-100, had a tightly bound Q, and thus two different ADHs, one having the tightly bound Q (Q-bound ADH) and Q-free ADH, could be obtained. The Q-binding sites of both the ADHs were characterized using specific inhibitors, a substituted phenol PC16 (a Q analog inhibitor) and antimycin A. Based on the inhibition kinetics of Q2 reductase and ubiquinol-2 (Q2H2) oxidase activities, it was suggested that there are one and two PC16-binding sites in Q-bound ADH and Q-free ADH respectively. On the other hand, with antimycin A, only one binding site was found for Q2 reductase and Q2H2 oxidase activities, irrespective of the presence of bound Q. These results suggest that ADH has a high-affinity Q binding site (QH) besides low-affinity Q reduction and QH2 oxidation sites, and that the bound Q in the QH site is involved in the electron transfer between heme c moieties and bulk Q or QH2 in the low-affinity sites.

Energy Metabolism of a Unique Acetic Acid Bacterium,Asaia bogorensis, That Lacks Ethanol Oxidation Activity

Acetic acid bacteria (AAB) are known as a vinegar producer on account of their ability to accumulate a high concentration of acetic acid due to oxidative fermentation linking the ethanol oxidation respiratory chain. Reactions in oxidative fermentation cause poor growth because a large amount of the carbon source is oxidized incompletely and the harmful oxidized products are accumulated almost stoichiometrically in the culture medium during growth, but a newly identified AAB, Asaia, has shown unusual properties, including scanty acetic acid production and rapid growth, as compared with known AAB as Acetobacter, Gluconobacter, and Gluconacetobacter. To understand these unique properties of Asaia in more detail, the respiratory chain and energetics of this strain were investigated. It was found that Asaia lacks quinoprotein alcohol dehydrogenase, but has other sugar and sugar alcohol-oxidizing enzymes specific to the respiratory chain of Gluconobacter, especially quinoprotein glycerol dehydrogenase. It was also found that Asaia has a cyanide-sensitive cytochrome bo(3)-type ubiquinol oxidase as sole terminal oxidase in the respiratory chain, and that it exhibits a higher H(+)/O ratio.

Disruption of quinoprotein ethanol dehydrogenase gene and adjacent genes in Pseudomonas putida HK5

Pseudomonas putida HK5 produces three different quinoprotein alcohol dehydrogenases: ADH-I, ADH-IIB and ADH-IIG. Gene organization of qedA, the gene for ADH-I, and other 10 genes in the cluster was related to the genome sequences of five other Pseudomonas strains. Insertion mutations in either qedA, exaE or agmR eliminated ADH-I activity, although the mutants were still able to grow on ethanol but more slowly than the wild-type strain. Mutant analysis demonstrated the requirement of agmR and exaE in ADH-I expression, and the tentative involvement of agmR, but not exaE, in the induction of ADH-IIB and ADH-IIG activities.

Genetic analyses and molecular characterization of the pathways involved in the conversion of 2‐phenylethylamine and 2‐phenylethanol into phenylacetic acid in Pseudomonas putida U

In Pseudomonas putida U two different pathways (Pea, Ped) are required for the conversion of 2-phenylethylamine and 2-phenylethanol into phenylacetic acid. The 2-phenylethylamine pathway (PeaABCDEFGHR) catalyses the transport of this amine, its deamination to phenylacetaldehyde by a quinohaemoprotein amine dehydrogenase and the oxidation of this compound through a reaction catalysed by a phenylacetaldehyde dehydrogenase. Another catabolic route (PedS(1)R(1)ABCS(2)R(2)DEFGHI) is needed for the uptake of 2-phenylethanol and for its oxidation to phenylacetic acid via phenylacetaldehyde. This implies the participation of two different two-component signal-transducing systems, two quinoprotein alcohol dehydrogenases, a cytochrome c, a periplasmic binding protein, an aldehyde dehydrogenase, a pentapeptide repeat protein and an ABC efflux system. Additionally, two accessory sets of elements (PqqABCDEF and CcmABCDEFGHI) are necessary for the operation of the main pathways (Pea and Ped). PqqABCDEF is required for the biosynthesis of pyrroloquinoline quinone (PQQ), a prosthetic group of certain alcohol dehydrogenases that transfers electrons to an independent cytochrome c; whereas CcmABCDEFGHI is required for cytochrome c maturation. Our data show that the degradation of phenylethylamine and phenylethanol in P. putida U is quite different from that reported in Escherichia coli, and they demonstrate that PeaABCDEFGHR and PedS(1)R(1)ABCS(2)R(2)DEFGHI are two upper routes belonging to the phenylacetyl-CoA catabolon.

Studies of Organic Protein Cofactors Using Electron Paramagnetic Resonance

Abstract The elucidation of structural and electronic aspects of paramagnetic organic cofactors in their protein surroundings and related model compounds is a topic of widespread interest. In this field, electron paramagnetic resonance (EPR) spectroscopy, especially in conjunction with high-resolution electron-nuclear double resonance (ENDOR) is an important tool, allowing us to analyze details of the distribution of the unpaired electron spin, which is influenced by the interaction between the cofactor and its immediate environment, as well as to characterize paramagnetic intermediate states in the course of the protein action. In this account, investigations are reported in which EPR/ENDOR methods have contributed significantly to answering questions on mechanistic and structural aspects of protein function. Emphasis is given to in-depth characterizations of electronic and spatial structures of radical cofactors and their positioning relative to substrate molecules. After a brief introduction, covering some of the trends of modern EPR/ENDOR method development, we will focus, as examples, on characterizations of quinone-type and flavin cofactors in two proteins, (i) quinoprotein alcohol dehydrogenase, and (ii) DNA photolyase.

Purification and Characterization of Membrane-Bound Quinoprotein Alcohol Dehydrogenase from a Native Strain of Acetbacter

Purification and Characterization of Membrane-Bound Quinoprotein Alcohol Dehydrogenase from a Native Strain of Acetbacter

Respiratory system of Gluconacetobacter diazotrophicus PAL5 Evidence for a cyanide-sensitive cytochrome bb and cyanide-resistant cytochrome ba quinol oxidases

In highly aerobic environments, Gluconacetobacter diazotrophicus uses a respiratory protection mechanism to preserve nitrogenase activity from deleterious oxygen. Here, the respiratory system was examined in order to ascertain the nature of the respiratory components, mainly of the cyanide sensitive and resistant pathways. The membranes of G. diazotrophicus contain Q 10, Q 9 and PQQ in a 13:1:6.6 molar ratios. UV 360 nm photoinactivation indicated that ubiquinone is the electron acceptor for the dehydrogenases of the outer and inner faces of the membrane. Strong inhibition by rotenone and capsaicin and resistance to flavone indicated that NADH-quinone oxidoreductase is a NDH-1 type enzyme. KCN-titration revealed the presence of at least two terminal oxidases that were highly sensitive and resistant to the inhibitor. Tetrachorohydroquinol was preferentially oxidized by the KCN-sensitive oxidase. Neither the quinoprotein alcohol dehydrogenase nor its associated cytochromes c were instrumental components of the cyanide resistant pathway. CO-difference spectrum and photodissociation of heme-CO compounds suggested the presence of cytochromes b-CO and a 1-CO adducts . Air-oxidation of cytochrome b (432 nm) was arrested by concentrations of KCN lower than 25 μM while cytochrome a 1 (442 nm) was not affected. A KCN-sensitive ( I 50 = 5 μM) cytochrome bb and a KCN-resistant ( I 50 = 450 μM) cytochrome ba quinol oxidases were separated by ion exchange chromatography.

Substrate binding in quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa studied by electron-nuclear double resonance

Binding of methanol to the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa has been studied by pulsed electron-nuclear double resonance at 9 GHz. Shifts in the hyperfine couplings of the pyrroloquinoline quinone radical provide direct evidence for a change in the environment of the cofactor when substrate is present. By performing experiments with deuteriated methanol, we confirmed that methanol was the cause of the effect. Density functional theory calculations show that these shifts can be understood if a water molecule, which is often found in x-ray structures of the active site of quinoprotein alcohol dehydrogenases, is displaced by the substrate. The difference between the binding of water and methanol is that the water molecule forms a hydrogen bond to O5 of pyrroloquinoline quinone, which the methanol, by virtue of its methyl group, does not. The results support the proposal that aspartate rather than glutamate is the catalytically active base for a hydride transfer mechanism in quinoprotein alcohol dehydrogenases.

Structure of the Pyrroloquinoline Quinone Radical in Quinoprotein Ethanol Dehydrogenase

Quinoprotein alcohol dehydrogenases use the pyrroloquinoline quinone (PQQ) cofactor to catalyze the oxidation of alcohols. The catalytic cycle is thought to involve a hydride transfer from the alcohol to the oxidized PQQ, resulting in the generation of aldehyde and reduced PQQ. Reoxidation of the cofactor by cytochrome proceeds in two sequential steps via the PQQ radical. We have used a combination of electron nuclear double resonance and density functional theory to show that the PQQ radical is not protonated at either O-4 or O-5, a result that is at variance with the general presumption of a singly protonated radical. The quantum mechanical calculations also show that reduced PQQ is unlikely to be protonated at O-5; rather, it is either singly protonated at O-4 or not protonated at either O-4 or O-5, a result that also challenges the common assumption of a reduced PQQ protonated at both O-4 and O-5. The reaction cycle of PQQ-dependent alcohol dehydrogenases is revised in light of these findings.

A Novel Type of Formaldehyde-Oxidizing Enzyme from the Membrane ofAcetobactersp. SKU 14

Membrane-bound NADP-independent formaldehyde-oxidizing enzyme was purified to homogeneity from the membrane of Acetobacter sp. SKU 14 isolated in Thailand. The enzyme was solubilized from the membrane fraction of glycerol-grown cells with 1% Tween 20 at pH 2.85, and purified to homogeneity through the steps of column chromatographies on DEAE-Sephadex A-50 and Q-Sepharose in the presence of 0.1% Tween 20 and 0.1% Triton X-100. The enzyme purified together with a cytochrome c showed a single protein band on native-PAGE, and was dissociated into three different subunits upon SDS-PAGE with molecular masses of 78 kDa, 55 kDa, and 18 kDa. The purified enzyme was finally characterized as a quinoprotein alcohol dehydrogenase (QADH), and this is the first indication that QADH highly oxidizes formaldehyde. The substrate specificity of the enzyme was found to be broad toward aldehydes and alcohols, and alcohols, especially n-butanol, n-propanol, and ethanol, were oxidized more rapidly than formaldehyde.