Information on EC 1.1.2.8 - alcohol dehydrogenase (cytochrome c)

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ethanol dehydrogenase

exaF

PedE

PedH

PpADH

PP_2674

PP_2679

PQQ-ADH

PQQ-alcohol dehydrogenase

PQQ-dependent alcohol dehydrogenases

PQQ-dependent type I alcohol dehydrogenase

pyrroloquinoline quinone ethanol dehydrogenase

pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenase

quinoprotein alcohol dehydrogenase

quinoprotein alcohol dehydrogenases

Biosynthesis of secondary metabolites, Chloroalkane and chloroalkene degradation, Glycolysis / Gluconeogenesis, Microbial metabolism in diverse environments, Pyruvate metabolism

a primary alcohol + 2 ferricytochrome c = an aldehyde + 2 ferrocytochrome c + 2 H+

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KEGG

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.

butanol + 2 cytochrome c

butyraldehyde + 2 reduced cytochrome c

diethylstilbestrol + pyrroloquinoline quinone

diethylstilbestrol 4-semiquinone + pyrroloquinoline quinol

ethanol + 2 cytochrome c

acetaldehyde + 2 reduced cytochrome c

ethanol + 2 cytochrome c

ethanal + 2 reduced cytochrome c

ethanol + 2 ferricytochrome c

ethanal + 2 ferrocytochrome c

ethanol + N,N,N',N'-tetramethyl-p-phenylenediamine

ethanal + ?

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i.e. Wurster's Blue

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?

ethanol + oxidized 2,6-dichlorophenolindophenol

ethanal + reduced 2,6-dichlorophenolindophenol

methanol + 2 cytochrome c

formaldehyde + 2 reduced cytochrome c

propanol + 2 cytochrome c

propanal + 2 reduced cytochrome c

additional information

?

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butanol + 2 cytochrome c

butyraldehyde + 2 reduced cytochrome c

diethylstilbestrol + pyrroloquinoline quinone

diethylstilbestrol 4-semiquinone + pyrroloquinoline quinol

ethanol + 2 cytochrome c

acetaldehyde + 2 reduced cytochrome c

ethanol + 2 ferricytochrome c

ethanal + 2 ferrocytochrome c

methanol + 2 cytochrome c

formaldehyde + 2 reduced cytochrome c

additional information

?

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cytochrome c

pyrroloquinoline quinone

12 entries

additional information

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Ca2+

5 entries

Iron

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one [2Fe-2S] cluster per enzyme

La3+

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required, active site-bound, 1.3 mol of La3+ per mol of ExaF protomer, indicating a 1:1 ratio of L3+ to protomer of enzyme

Mg2+

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required

Sr2+

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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.

701600

additional information

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the enzyme from strain AM1 utilizes La3+ rather than Ca2+ as a cofactor

ethylamine

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activating at low concentrations, inhibitory at high concentrations

pentylamine

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the alcohol oxidation activity of PpADH is strongly enhanced by pentylamine and the affinity for substrates is also improved by pentylamine as an activator, inhibitory at high concentrations

ammonium

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Butylamine

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Dimethylamine

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dipropylamine

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ethanol

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does not affect the adhS gene expression but induces PQQ-ADH activity

ethylamine

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inhibitory at high concentrations

heptylamine

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Hexylamine

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Isobutylamine

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slight activation

methylamine

octylamine

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pentylamine

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the alcohol oxidation activity of PpADH is strongly enhanced by pentylamine and the affinity for substrates is also improved by pentylamine as an activator, inhibitory at high concentrations

Propylamine

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triethylamine

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trimethylamine

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tripropylamine

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additional information

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the enzyme requires ammonia or primary amines as activators in in vitro assays with artificial electron acceptors, the enzyme is activated by various primary amines, dimethylamine, and trimethylamine. tert-Butylamine is a poor activator. The hydrophobic interaction contributes to the binding of amine activators to the binding site in the enzyme

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0.14 - 0.21

ethanol

0.35 - 0.43

Propanol

24.8 - 56.6

ethanol

32.6 - 73.1

Propanol

115 - 395

ethanol

73 - 209

Propanol

112.5

ethylamine

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pH 9.0, temperature not specified in the publication

37.6

pentylamine

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pH 9.0, temperature not specified in the publication

40

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pH 9.0, 25°C

6

C7G3B7, C7G3B8, C9K501, C9K502, C9K504, C9W9E3, D2SZY4, D2SZY5

assay at

7.5

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assay at

9

25

30

35

C7G3B7, C7G3B8, C9K501, C9K502, C9K504, C9W9E3, D2SZY4, D2SZY5

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6.77

Pseudomonas mendocina

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sequence calculation

6.96

Pseudomonas stutzeri

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sequence calculation

Acetobacter pasteurianus IFO3191

9 entries

Acetobacter pasteurianus MSU10

Acetobacter pasteurianus SKU1108

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Gluconacetobacter diazotrophicus PAL5

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-

-

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Methylorubrum extorquens ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1

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Pseudomonas mendocina

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Pseudomonas mendocina YMP

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-

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Pseudomonas putida KT 2240

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-

-

-

-

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Pseudomonas stutzeri ATCC 14405

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-

additional information

evolution

malfunction

metabolism

physiological function

12 entries

additional information

Methylacidiphilum fumariolicum SolV

Pseudogluconobacter saccharoketogenes

Pseudomonas putida KT2440

115000

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inactive enzyme form, native PAGE

120000

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inactive enzyme form, gel filtration

126400

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recombinant His6-tagged enzyme, gel filtration

136000

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gel filtration

330000

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active enzyme form, gel filtration

345000

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active enzyme form, native PAGE

45000

60000

72000

9000

?

dimer

1 * 60000 + 1 * 9000, SDS-PAGE

heterodimer

heterotetramer

homodimer

tetramer

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2 * 60000, alpha-subunit, + 2 * 9000, beta-subunit, SDS-PAGE

additional information

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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

additional information

sequence contains a signal peptide of 34 residues

389964

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

389964

diffraction to beyond 2.5 A, space group R3

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705106

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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389959

A26V

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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability

G55D

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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability

L18Q

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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability

T104K

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site-directed mutagenesis, the mutation leads to a complete loss of ethanol oxidizing ability

V107A

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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability

V36I

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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability

V54I

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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability

V70A

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site-directed mutagenesis, the mutation does not affect the PQQ-ADH activity and ethanol oxidizing ability

C105A/C106A

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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

674560

additional information

10 entries

acetic acid

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native enzyme from membranes by two steps of anion exchange chormatography, hydroxyapatite chromatography, and gel filtration

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native enzyme from strain IFO3191 by ultracentrifugation and anion exchange chromatography

C7G3B7, C7G3B8, C9K501, C9K502, C9K504, C9W9E3, D2SZY4, D2SZY5

native enzyme from strain MSU10 by ultracentrifugation and anion exchange chromatography

C7G3B7, C7G3B8, C9K501, C9K502, C9K504, C9W9E3, D2SZY4, D2SZY5

native enzyme from strain SKU1108 by ultracentrifugation and anion exchange chromatography

C7G3B7, C7G3B8, C9K501, C9K502, C9K504, C9W9E3, D2SZY4, D2SZY5

recombinant His6-tagged enzyme from Escherichia coli strain TOP10 by ultracentrifugation, nickel affinity chromatography, ultrafiltration, and desalting gel filtration

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389964

gene adhA, DNA and amino acid sequence determination and analysis, sequence comparison of the genes encoding subunit I, subunit II, and subunit III of ADHs

C7G3B7, C7G3B8, C9K501, C9K502, C9K504, C9W9E3, D2SZY4, D2SZY5

gene adhB, DNA and amino acid sequence determination and analysis, sequence comparison of the genes encoding subunit I, subunit II, and subunit III of ADHs

C7G3B7, C7G3B8, C9K501, C9K502, C9K504, C9W9E3, D2SZY4, D2SZY5

gene adhS, DNA and amino acid sequence determination and analysis, sequence comparison of the genes encoding subunit I, subunit II, and subunit III of ADHs

C7G3B7, C7G3B8, C9K501, C9K502, C9K504, C9W9E3, D2SZY4, D2SZY5

gene exaF, or exaA, DNA and amino acid sequence determination and analysis, functional recombinant expression of C-terminally His6-tagged enzyme in Escherichia coli strain TOP10

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sequence comparisons and phylogenetic tree, real-time reverse transcriptase PCR expression analysis

ethanol does not affect the adhS gene expression but induces PQQ-ADH activity

the enzyme is induced by diethylstilbestrol, presence of diethylstilbestrol causes a 5.2fold increase in the mRNA level of the gene encoding quinoprotein alcohol dehydrogenase

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.