Quinoprotein Ethanol Dehydrogenase Research Articles

Pyrroloquinoline Quinone Ethanol Dehydrogenase in Methylobacterium extorquens AM1 Extends Lanthanide-Dependent Metabolism to Multicarbon Substrates.

ExaF is the most efficient PQQ-dependent ethanol dehydrogenase reported to date and, to our knowledge, the first non-XoxF-type alcohol oxidation system reported to use lanthanides as a cofactor, expanding the importance of lanthanides in biochemistry and bacterial metabolism beyond methanol dehydrogenases to multicarbon metabolism. These results support an earlier proposal that an aspartate residue near the catalytic aspartate residue may be an indicator of rare-earth element utilization by type I alcohol dehydrogenases.

Quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: the unusual disulfide ring formed by adjacent cysteine residues is essential for efficient electron transfer to cytochrome c 550

All pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases contain an unusual disulfide ring formed between adjacent cysteine residues. A mutant enzyme that is lacking this structure was generated by replacing Cys105 and Cys106 with Ala in quinoprotein ethanol dehydrogenase (QEDH) from Pseudomonas aeruginosa ATCC17933. Heterologously expressed quinoprotein ethanol dehydrogenase in which Cys-105 and Cys-106 have been replaced by Ala (Cys105Ala/Cys106Ala apo-QEDH) was successfully converted to enzymatic active holo-enzyme by incorporation of its cofactor PQQ in the presence of Ca(2+). The enzymatic activity of the mutant enzyme in the artificial dye test with N-methylphenazonium methyl sulfate (PMS) and 2,6-dichlorophenol indophenol (DCPIP) at pH 9 did not depend on an activating amine which is essential for wild type activity under these conditions. The mutant enzyme showed increased Michaelis constants for primary alcohols, while the affinity for the secondary alcohol 2-propanol was unaltered. Surprisingly, for all substrates tested the specific activity of the mutant enzyme in the artificial dye test was higher than that found for wild type QEDH. On the contrary, in the ferricyanide test with the natural electron acceptor cytochrome c(550) the activity of mutant Cys105Ala/Cys106Ala was 15-fold lower than that of wild type QEDH. We demonstrate for the first time unambiguously that the unusual disulfide ring is essential for efficient electron transfer at pH 7 from QEDH to its natural electron acceptor cytochrome c(550).

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.

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.

Substrate-Binding in Quinoprotein Ethanol Dehydrogenase from Pseudomonas aeruginosa Studied by Electron Paramagnetic Resonance at 94 GHz

Pyrroloquinoline quinone (2,7,9-tricarboxypyrroloquinoline quinone, PQQ) is one of several quinone cofactors that is utilized in a class of dehydrogenases known as quinoproteins. In this contribution, we have used continuous-wave high-field/high-frequency electron paramagnetic resonance (EPR) at 94 GHz (W-band) to study substrate binding in ethanol dehydrogenase (QEDH) from Pseudomonas aeruginosa, taking advantage of the fact that the enzyme is isolated with a substantial proportion of the PQQ cofactor in the paramagnetic semiquinone form. In the substrate-free enzyme, the principal values of the g-tensor, obtained by spectral simulation are: gx = 2.00585(2), gy = 2.00518(2), and gz = 2.00212(2), giving giso = 2.00438(2). All three principal values of the g-tensor decrease when ethanol is bound to the protein: gx = 2.00574(2), gy = 2.00511(2), and gz = 2.00207(2), giving giso = 2.00431(2). The results represent the first direct evidence for the tight binding of an alcohol to a PQQ-dependent alcohol dehydrogenase and show that ethanol also binds to the enzyme even when the PQQ cofactor is in the semiquinone form. The decrease in g is consistent with an increase in polarity in the immediate vicinity of the PQQ cofactor and probably reflects a changed geometry of the PQQ-Ca2+ complex when ethanol binds.

Characterisation of the PQQ cofactor radical in quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa by electron paramagnetic resonance spectroscopy

The binding pocket of the pyrroloquinoline quinone (PQQ) cofactor in quinoprotein alcohol dehydrogenases contains a characteristic disulphide ring formed by two adjacent cysteine residues. To analyse the function of this unusual structural motif we have investigated the wild-type and a double cysteine:alanine mutant of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa by electron paramagnetic resonance (EPR) spectroscopy. Thus, we have obtained the principal values for the full rhombic g-tensor of the PQQ semiquinone radical by high-field (94 GHz) EPR necessary for a discrimination of radical species in dehydrogenases containing PQQ together with other redox-active cofactors. Our results show that the characteristic disulphide ring is no prerequisite for the formation of the functionally important semiquinone form of PQQ.

A soluble two-component regulatory system controls expression of quinoprotein ethanol dehydrogenase (QEDH) but not expression of cytochrome c(550) of the ethanol-oxidation system in Pseudomonas aeruginosa.

The regulation of the divergent promoters of the exaAB genes in Pseudomonas aeruginosa ATCC 17933, in which exaA encodes a quinoprotein ethanol dehydrogenase and exaB codes for a cytochrome c(550), was studied. Using transcriptional lacZ fusions, promoter activity during growth on several substrates was measured. These promoter-probe vectors were also used to identify regulatory mutants defective in exaAB induction. Transcription from both exaA and exaB was reduced significantly in four mutants. Two other mutants showed transcription from exaA that was reduced, but higher than wild-type transcription from exaB. The genes that are needed for exaA promoter induction were sequenced and found to encode a two-component regulatory system: a histidine sensor kinase, which lacks a transmembrane helical N-terminus and is presumably located in the cytoplasm, and a response regulator. The phenotypic characterization and restoration of the wild-type behaviour of the different regulatory mutants produced by different cosmids and subclones indicate that six different genes may be involved in regulating ethanol oxidation in P. aeruginosa.

X-ray structure of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: basis of substrate specificity

The homodimeric enzyme form of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa ATCC 17933 crystallizes readily with the space group R3. The X-ray structure was solved at 2.6 Å resolution by molecular replacement.Aside from differences in some loops, the folding of the enzyme is very similar to the large subunit of the quinoprotein methanol dehydrogenases from Methylobacterium extorquens or Methylophilus W3A1. Eight W-shaped β-sheet motifs are arranged circularly in a propeller-like fashion forming a disk-shaped superbarrel. No electron density for a small subunit like that in methanol dehydrogenase could be found. The prosthetic group is located in the centre of the superbarrel and is coordinated to a calcium ion. Most amino acid residues found in close contact with the prosthetic group pyrroloquinoline quinone and the Ca2+ are conserved between the quinoprotein ethanol dehydrogenase structure and that of the methanol dehydrogenases. The main differences in the active-site region are a bulky tryptophan residue in the active-site cavity of methanol dehydrogenase, which is replaced by a phenylalanine and a leucine side-chain in the ethanol dehydrogenase structure and a leucine residue right above the pyrrolquinoline quinone group in methanol dehydrogenase which is replaced by a tryptophan side-chain. Both amino acid exchanges appear to have an important influence, causing different substrate specificities of these otherwise very similar enzymes. In addition to the Ca2+ in the active-site cavity found also in methanol dehydrogenase, ethanol dehydrogenase contains a second Ca2+-binding site at the N terminus, which contributes to the stability of the native enzyme.

Cytochrome c550 is an essential component of the quinoprotein ethanol oxidation system in Pseudomonas aeruginosa: cloning and sequencing of the genes encoding cytochrome c550 and an adjacent acetaldehyde dehydrogenase.

Pseudomonas aeruginosa ATCC 17933 grown aerobically on ethanol produces a soluble cytochrome c550 together with a quinoprotein ethanol dehydrogenase. A 3.2 kb genomic DNA fragment containing the gene encoding cytochrome c550 was cloned and sequenced. Two other complete and two truncated ORFs were also identified. A truncated ORF encoding the quinoprotein ethanol dehydrogenase (exaA) was found upstream of the cytochrome c550 gene (exaB) and in reverse orientation. An ORF encoding a NAD(+)-dependent acetaldehyde dehydrogenase (exaC) was located downstream of the cytochrome c550 gene and in the same orientation. Another ORF showed similarity to the pqqA gene and a truncated ORF similarity to the pqqB gene, both involved in the biosynthesis of the prosthetic group PQQ. The organization of these genes was found to be different from the well-studied methanol oxidation system in methylotrophic bacteria. The deduced amino acid sequence of cytochrome c550 from P. aeruginosa showed some similarity to cytochrome c6 of the alga Chlamydomonas reinhardtii and the haem domain of quinohaemoprotein alcohol dehydrogenases of acetic acid bacteria, but no similarity to the soluble cytochrome cL of the quinoprotein methanol oxidation system of methylotrophs could be detected. A mutant of P. aeruginosa with an interrupted cytochrome c550 gene was unable to grow on ethanol, which proves that cytochrome c550 is an essential component of the ethanol oxidation system in this organism.

Quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa is a homodimer--sequence of the gene and deduced structural properties of the enzyme.

The gene coding for the periplasmic quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa ATCC 17933 was cloned and sequenced. The deduced amino acid sequence contained a signal peptide of 34 residues and the major protein of 589 amino acids showed high similarities to pyrroloquinoline-quinone-dependent periplasmic and membrane-bound dehydrogenases acting on alcohols, glucose and quinate or shikimate. It was demonstrated by alignment with the amino acid sequence of the large subunit of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens, whose X-ray structure is known, that the amino acid residues involved in the binding of pyrroloquinoline quinone and Ca2+ at the active site are conserved in the quinoprotein ethanol dehydrogenase of P. aeruginosa. Also, the glycine/tryptophan docking motifs involved in stabilizing the superbarrel structure of the quinoprotein methanol dehydrogenase of M. extorquens were conserved. The known sequences of pyrroloquinoline-quinone-dependent dehydrogenases were used to derive new, more specific sequence motifs for detecting members of this family of enzymes. Despite the sequence similarity between the large a subunit of quinoprotein methanol dehydrogenase from M. extorquens and the quinoprotein ethanol dehydrogenase from P. aeruginosa, the two enzyme systems were quite different. In the presence of the prosthetic group, pyrroloquinoline quinone expression of the Pseudomonas gene encoding the 60-kDa subunit of quinoprotein ethanol dehydrogenase in Escherichia coli resulted in formation of active enzyme. The formation of active quinoprotein methanol dehydrogenase, however, is known to require, in addition to the large alpha subunit, the expression of a small beta subunit, and helper proteins [Lidstrom, M. E. (1995) Genetics of bacterial quinoproteins, Methods Enzymol. 258, 217-227].

Quaternary structure of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa and its reoxidation with a novel cytochrome c from this organism

Quinoprotein (2,7,9-tricarboxy-1H-pyrrolo-[2,3-f]quinoline-4,5-dione quinone form (PQQ)-containing) ethanol dehydrogenase (EDH) from Pseudomonas aeruginosa ATCC 17933 was purified to homogeneity. EDH has an alpha 2 beta 2 configuration and subunits comparable in size to those of methanol dehydrogenase (MDH). Compared with other PQQ-containing dehydrogenases, Ca2+ is rather loosely bound and it seems necessary for PQQ binding and stability of EDH. Two soluble cytochromes c were detected in extracts from ethanol-grown cells and both were purified. One of these has an alpha-band at 551 nm for its reduced form, the oxidized form being an excellent electron acceptor for the semiquinone form of EDH. Since this cytochrome is quite different from the already known cytochrome c551 (operating in nitrate respiration) of this organism, it is indicated here as cytochrome cEDH. Comparison of the N-terminal amino acid sequence of cytochrome cEDH with the complete sequence of cytochrome cL (the electron acceptor of MDH), cytochrome cH (the electron acceptor of cytochrome cL) and cytochrome c551 revealed some similarity only to internal stretches of amino acids of the last two. The other soluble cytochrome appeared to be the already-known cytochrome c556. Since it was not an electron acceptor for cytochrome cEDH (neither for EDH), cytochrome cH is lacking in the quinoprotein-EDH-ethanol oxidation system of P. aeruginosa. It seems, therefore, that the respiratory chains for MDH and EDH are different.

Cytochrome c550 from Pseudomonas aeruginosa

In cells of Pseudomonas aeruginosa A.T.C.C. 17933 grown on ethanol the synthesis of a soluble c-type cytochrome, together with quinoprotein ethanol dehydrogenase, is induced. The cytochrome, with an alpha-absorption band at 550 nm, was purified to homogeneity. The molecular mass of the monomeric protein is 15 kDa, the pI is 4.8, and it contains one haem prosthetic group. The midpoint potential of the autoxidizable, but not autoreducible, cytochrome is 280 mV. Cytochrome c550 mediates electron transfer between quinoprotein ethanol dehydrogenase and ferricyanide. In a system composed of membrane particles with NN'NN'-tetramethyl-p-phenylenediamine oxidase activity and quinoprotein ethanol dehydrogenase, oxygen consumption is only observed in the presence of cytochrome c550. This indicates the participation of the cytochrome in the electron-transport chain linked to quinoprotein ethanol dehydrogenase in P. aeruginosa. The electron transport from ethanol dehydrogenase to oxygen is inhibited by myxothiazol and antimycin, indicating that a cytochrome bc1-like complex is involved.

Quinoprotein Ethanol Dehydrogenase:Preparation of the Apo-form and Reconstitution with Pyrroloquinoline Quinone and Ca2+ or Sr2+ Ions.

Homogeneous quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa ATCC 17933 contains one Ca2+ ion per subunit of native enzyme. Treatment with fra/is-1,2-diaminocyclohexane-N,N,N,N′N′-tetraacetic acid at 30°C led to an catalytically inactive apo-form. Upon incubation of the apo-form with Ca2+ and pyrroloquinoline quinone a fully active holo-enzyme was reconstituted. The absorption spectrum of this reconstituted enzyme is identical to that of the native enzyme. Incubation of apo-enzyme with Sr2+ and PQQ led to the formation of an active Sr2+-form. The Sr2+ and the Ca2+-forms of the enzyme differ in their absorption spectra. The Sr2+-form was inactivated by frawv-1,2-diaminocyclohexane-N,N,N,N′N′-tetraacetic acid twice as fast as the Ca2+-form. Ca2+ is necessary for pyrroloquinoline quinone to bind to the apo-form of quinoprotein ethanol dehydrogenase.

Quinoprotein ethanol dehydrogenase from Pseudomonas.

Dye-linked ethanol dehydrogenases from Pseudomonas aeruginosa ATCC 17,933 and P. putida ATCC 17,421 were purified to homogeneity and crystallized. The amino acid composition of the two enzymes is very similar and the number of the aromatic amino acid residues found per subunit are almost identical. With respect to their catalytic and molecular properties both ethanol dehydrogenases are similar to the quinoprotein methanol dehydrogenases known from methylotrophic bacteria. They show a high pH-optimum, need ammonia or an amine as activator and are dimers of identical subunits of a molecular mass of 60,000. The dimer is the catalytically active form. Each subunit carries one prosthetic group pyrroloquinoline quinone, which can be titrated by the suicide substrate cyclopropanone ethylhemiketal. In contrast to the general methanol dehydrogenases the two ethanol dehydrogenases have a low affinity for methanol and in addition to primary alcohols they also oxidize secondary alcohols. With secondary alcohols preferentially one of the two enantiomers is oxidized. The catalytic and spectral properties of the two enzymes are very similar to the quinoprotein ethanol dehydrogenase isolated from P. aeruginosa LMD 80.53 (Groen et al., 1984. Biochem. J. 223: 921-924). However this enzyme is reported to be a monomer of molecular mass 100,000.

Preliminary X-ray crystallographic study of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa

The homodimeric enzyme form of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa ATCC 17933 crystallizes readily with the space group R3. The X-ray structure was solved at 2.6 Å resolution by molecular replacement.Aside from differences in some loops, the folding of the enzyme is very similar to the large subunit of the quinoprotein methanol dehydrogenases from Methylobacterium extorquens or Methylophilus W3A1. Eight W-shaped β-sheet motifs are arranged circularly in a propeller-like fashion forming a disk-shaped superbarrel. No electron density for a small subunit like that in methanol dehydrogenase could be found. The prosthetic group is located in the centre of the superbarrel and is coordinated to a calcium ion. Most amino acid residues found in close contact with the prosthetic group pyrroloquinoline quinone and the Ca2+ are conserved between the quinoprotein ethanol dehydrogenase structure and that of the methanol dehydrogenases. The main differences in the active-site region are a bulky tryptophan residue in the active-site cavity of methanol dehydrogenase, which is replaced by a phenylalanine and a leucine side-chain in the ethanol dehydrogenase structure and a leucine residue right above the pyrrolquinoline quinone group in methanol dehydrogenase which is replaced by a tryptophan side-chain. Both amino acid exchanges appear to have an important influence, causing different substrate specificities of these otherwise very similar enzymes. In addition to the Ca2+ in the active-site cavity found also in methanol dehydrogenase, ethanol dehydrogenase contains a second Ca2+-binding site at the N terminus, which contributes to the stability of the native enzyme.

Purification, crystallisation and characterization of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa ATCC 17933 when grown on ethanol produces high levels of a quinoprotein ethanol dehydrogenase, which amounts to 7% of the soluble protein. The enzyme has been purified to homogeneity and it crystallizes readily in the presence of polyethylene glycol 1550 or 6000. The ethanol dehydrogenase (Km(ethanol) = 14 microM) resembles the dye-dependent quinoprotein methanol dehydrogenases of methylotrophic bacteria, but has a low affinity for methanol (Km (methanol) = 94mM). In addition the enzyme oxidizes secondary alcohols. With its catalytic properties the ethanol dehydrogenase is similar to the enzyme isolated from P. aeruginosa LMD 80.53 (Groen, B., Frank, J. Jzn. & Duine, J.A. (1984) Biochem. J. 223, 921-924). In contrast to this enzyme from P. aeruginosa LMD 80.53, which is a monomer, the ethanol dehydrogenase isolated from P. aeruginosa ATCC 17933 is a dimer of identical subunits of relative molecular mass 60,000. The N-terminal amino acid is lysine. Inactivation with cyclopropanone ethylhemiketal reveals one molecule of pyrroloquinoline quinone per subunit. As shown by active enzyme sedimentation, the dimer is the enzymatically active form.