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Information on EC 1.1.2.8 - alcohol dehydrogenase (cytochrome c) and Organism(s) Pseudomonas aeruginosa and UniProt Accession Q9Z4J7

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IUBMB Comments
A periplasmic PQQ-containing quinoprotein. Occurs in Pseudomonas and Rhodopseudomonas. The enzyme from Pseudomonas aeruginosa uses a specific inducible cytochrome c550 as electron acceptor. Acts on a wide range of primary and secondary alcohols, but not methanol. It has a homodimeric structure [contrasting with the heterotetrameric structure of EC 1.1.2.7, methanol dehydrogenase (cytochrome c)]. It is routinely assayed with phenazine methosulfate as electron acceptor. Activity is stimulated by ammonia or amines. Like all other quinoprotein alcohol dehydrogenases it has an 8-bladed 'propeller' structure, a calcium ion bound to the PQQ in the active site and an unusual disulfide ring structure in close proximity to the PQQ.
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Pseudomonas aeruginosa
UNIPROT: Q9Z4J7
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The taxonomic range for the selected organisms is: Pseudomonas aeruginosa
The expected taxonomic range for this enzyme is: Bacteria, Archaea
Synonyms
ppadh, pqq-dh9, pyrroloquinoline quinone-dependent alcohol dehydrogenases, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SYSTEMATIC NAME
IUBMB Comments
alcohol:cytochrome c oxidoreductase
A periplasmic PQQ-containing quinoprotein. Occurs in Pseudomonas and Rhodopseudomonas. The enzyme from Pseudomonas aeruginosa uses a specific inducible cytochrome c550 as electron acceptor. Acts on a wide range of primary and secondary alcohols, but not methanol. It has a homodimeric structure [contrasting with the heterotetrameric structure of EC 1.1.2.7, methanol dehydrogenase (cytochrome c)]. It is routinely assayed with phenazine methosulfate as electron acceptor. Activity is stimulated by ammonia or amines. Like all other quinoprotein alcohol dehydrogenases it has an 8-bladed 'propeller' structure, a calcium ion bound to the PQQ in the active site and an unusual disulfide ring structure in close proximity to the PQQ.
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ethanol + oxidized 2,6-dichlorophenolindophenol
ethanal + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
-
-
-
?
ethanol + 2 cytochrome c
ethanal + 2 reduced cytochrome c
show the reaction diagram
-
EPR-study to elucidate reaction mechanism. In an addition/elimination mechanism, the negatively charged substrate oxygen then performs a nucleophilic addition to the PQQ(C5) to form a covalent substrate-PQQ complex. This is followed by elimination of ethanal, leaving the fully reduced PQQH2. In a hydride transfer mechanism, a nucleophilic addition to the PQQ(C5) again occurs, but this time it is the hydride from C1 of the substrate that is transferred, completing the oxidization of the ethanol to ethanal. Subsequently, the PQQ enolizes to form PQQH2. The results are consistent with either proposed mechanism
-
-
?
ethanol + N,N,N',N'-tetramethyl-p-phenylenediamine
ethanal + ?
show the reaction diagram
-
i.e. Wurster's Blue
-
-
?
ethanol + oxidized 2,6-dichlorophenolindophenol
ethanal + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
-
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
pyrroloquinoline quinone
-
pyrroloquinoline quinone
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Sr2+
-
incubation of apo-enzyme with Sr2+ and pyrroloquinoline quinone leads 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 is inactivated by trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid twice as fast as the Ca2+-form.
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
-
pH 9.0, 25°C
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
large subunit
UniProt
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60000
1 * 60000 + 1 * 9000, SDS-PAGE
9000
1 * 60000 + 1 * 9000, SDS-PAGE
136000
-
gel filtration
60000
-
2 * 60000, alpha-subunit, + 2 * 9000, beta-subunit, SDS-PAGE
9000
-
2 * 60000, alpha-subunit, + 2 * 9000, beta-subunit, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
1 * 60000 + 1 * 9000, SDS-PAGE
tetramer
-
2 * 60000, alpha-subunit, + 2 * 9000, beta-subunit, SDS-PAGE
additional information
-
enzyme interacts with a soluble cytochrome cEDH, the oxidized form being an excellent acceptor for the semiquinone form of EDH. This cytochrome is quite different from the cytochrome c551 operating in nitrate respiration
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
sequence contains a signal peptide of 34 residues
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
alignment with the amino acid sequence of the large subunit of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens. The amino acid residues involved in the binding of pyrroloquinoline quinone and Ca2+ at the active site are conserved
diffraction to beyond 2.5 A, space group R3
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to 2.6 A resolution, by molecular replacement. Eight W-shaped beta-sheet motifs are arranged circularly in a propeller-like fashion forming a disk-shaped superbarrel. 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 from Methylobacterium extorquens or Methylophilus W3A1. 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 contribute to different substrate specificities of these otherwise very similar enzymes. In addition to the Ca2+ in the active-site cavity, ethanol dehydrogenase contains a second Ca2+-binding site at the N-terminus, which contributes to the stability of the native enzyme
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C105A/C106A
-
mutation of residues forming a characteristic disulfide ring in the binding pocket of pyrroloquinoline quinone. Analysis by EPR spectroscopy shows that the disulfide ring is no prerequisite for the formation of the functionally important semiquinone form of pyrroloquinoline quinone
additional information
In the presence of the prosthetic group, expression of the Pseudomonas gene encoding the 60-kDa subunit of quinoprotein ethanol dehydrogenase in Escherichia coli results in formation of active enzyme
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
treatment with trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid at 30°C leads to an catalytically inactive apo-form. Upon incubation of the apo-form with Ca2+ and pyrroloquinoline quinone a fully active holo-enzyme is reconstituted. Incubation of apo-enzyme with Sr2+ and pyrroloquinoline quinone leads to the formation of an active Sr2+-form. The Sr2+ and the Ca2+-forms of the enzyme differ in their absorption spectra.
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Keitel, T.; Diehl, A.; Knaute, T.; Stezowski, J.J.; Hhne, W.; Grisch, H.
X-ray structure of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: basis of substrate specificity
J. Mol. Biol.
297
961-974
2000
Pseudomonas aeruginosa
Manually annotated by BRENDA team
Diehl, A.; v.Wintzingerode, F.; Grisch, H.
Quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa is a homodimer. Sequence of the gene and deduced structural properties of the enzyme
Eur. J. Biochem.
257
409-419
1998
Pseudomonas aeruginosa (Q9Z4J7)
Manually annotated by BRENDA team
Kay, C.W.; Mennenga, B.; Goerisch, H.; Bittl, R.
Structure of the pyrroloquinoline quinone radical in quinoprotein ethanol dehydrogenase
J. Biol. Chem.
281
1470-1476
2006
Pseudomonas aeruginosa
Manually annotated by BRENDA team
Kay, C.W.; Mennenga, B.; Goerisch, H.; Bittl, R.
Substrate binding in quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa studied by electron-nuclear double resonance
Proc. Natl. Acad. Sci. USA
103
5267-5272
2006
Pseudomonas aeruginosa
Manually annotated by BRENDA team
Mutzel, A.; Goerisch, H.
Quinoprotein ethanol dehydrogenase: preparation of the apo-form and reconstitution with pyrroloquinoline quinone and calcium or strontium(2+) ions
Agric. Biol. Chem.
55
1721-1726
1991
Pseudomonas aeruginosa
-
Manually annotated by BRENDA team
Schrover, J.M.; Frank, J.; van Wielink, J.E.; Duine, J.A.
Quaternary structure of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa and its reoxidation with a novel cytochrome c from this organism
Biochem. J.
290 (Pt 1)
123-127
1993
Pseudomonas aeruginosa
Manually annotated by BRENDA team
Stezowski, J.J.; Gorisch, H.; Dauter, Z.; Rupp, M.; Hoh, A.; Englmaier, R.; Wilson, K.
Preliminary X-ray crystallographic study of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa
J. Mol. Biol.
205
617-618
1989
Pseudomonas aeruginosa
Manually annotated by BRENDA team