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Information on EC 1.1.9.1 - alcohol dehydrogenase (azurin) and Organism(s) Comamonas testosteroni and UniProt Accession Q46444
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1.1.9.1 alcohol dehydrogenase (azurin)IUBMB Comments
A soluble, periplasmic PQQ-containing quinohemoprotein. Also contains a single heme c. Occurs in Comamonas and Pseudomonas. Does not require an amine activator. Oxidizes a wide range of primary and secondary alcohols, and also aldehydes and large substrates such as sterols; methanol is not a substrate. Usually assayed with phenazine methosulfate or ferricyanide. 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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The taxonomic range for the selected organisms is: Comamonas testosteroni
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
ADH IIB, ADH IIG, EC 1.1.98.1, QH-ADH, quinohaemoprotein alcohol dehydrogenase, quinohemoprotein alcohol dehydrogenase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
SYSTEMATIC NAME
IUBMB Comments
alcohol:azurin oxidoreductase
A soluble, periplasmic PQQ-containing quinohemoprotein. Also contains a single heme c. Occurs in Comamonas and Pseudomonas. Does not require an amine activator. Oxidizes a wide range of primary and secondary alcohols, and also aldehydes and large substrates such as sterols; methanol is not a substrate. Usually assayed with phenazine methosulfate or ferricyanide. 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)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(R)-2-butanol + ferricyanide
butan-2-one + ferrocyanide
-
enzyme electrode, 6.5% of vmax with 1-butanol, soluble enzyme, 6% of vmax with 1-butanol. With enzyme electrode, (S)-enantiomer is 13fold preferred over (R)-enantiomer, with soluble enzyme 13.5fold, respectively
-
-
?
(R)-2-heptanol + ferricyanide
heptan-2-one + ferrocyanide
-
enzyme electrode, 10% of vmax with 1-butanol, soluble enzyme, 5.5% of vmax with 1-butanol. With enzyme electrode, (S)-enantiomer is 82fold preferred over (R)-enantiomer, with soluble enzyme 315fold, respectively
-
-
?
(R)-2-hexanol + ferrocyanide
hexan-2-one + ferricyanide
-
enzyme electrode, 14% of vmax with 1-butanol, soluble enzyme, 9% of vmax with 1-butanol. With enzyme electrode, (S)-enantiomer is 35fold preferred over (R)-enantiomer, with soluble enzyme 105fold, respectively
-
-
?
(R)-2-octanol + ferrocyanide
octan-2-one + ferricyanide
-
enzyme electrode, 8% of vmax with 1-butanol. With enzyme electrode, (S)-enantiomer is 82fold preferred over (R)-enantiomer, with soluble enzyme 800fold, respectively
-
-
?
(R)-3-bromo-2-methyl-1-propanol + ferricyanide
(R)-3-bromo-2-methyl-propanal + ferrocyanide
-
-
-
-
r
(R)-glycidol + ferrocyanide
? + ferricyanide
-
enzyme electrode, 37% of vmax with 1-butanol, soluble enzyme, 1.6% of vmax with 1-butanol. With enzyme electrode, (S)-enantiomer is 0.9fold preferred over (R)-enantiomer, with soluble enzyme no distinction
-
-
?
(R)-solketal + 2 ferricyanide
(R)-solketal aldehyde + 2 ferrocyanide + 2 H+
-
(R)-enantiomer is prefered, enantiomeric ratio is 30
-
-
r
(R)-solketal + ferrocyanide
? + ferricyanide
-
enzyme electrode, 35% of vmax with 1-butanol, soluble enzyme, 10% of vmax with 1-butanol. With enzyme electrode, (S)-enantiomer is 39fold preferred over (R)-enantiomer, with soluble enzyme 117fold, respectively
-
-
?
(S)-2-butanol + ferricyanide
butan-2-one + ferrocyanide
-
enzyme electrode, 14% of vmax with 1-butanol, soluble enzyme, 13% of vmax with 1-butanol. With enzyme electrode, (S)-enantiomer is 13fold preferred over (R)-enantiomer, with soluble enzyme 13.5fold, respectively
-
-
?
(S)-2-heptanol + ferricyanide
heptan-2-one + ferrocyanide
-
enzyme electrode, 70% of vmax with 1-butanol, soluble enzyme, 63% of vmax with 1-butanol. With enzyme electrode, (S)-enantiomer is 82fold preferred over (R)-enantiomer, with soluble enzyme 315fold, respectively
-
-
?
(S)-2-hexanol + ferrocyanide
hexan-2-one + ferricyanide
-
enzyme electrode, 74% of vmax with 1-butanol, soluble enzyme, 71% of vmax with 1-butanol. With enzyme electrode, (S)-enantiomer is 35fold preferred over (R)-enantiomer, with soluble enzyme 105fold, respectively
-
-
?
(S)-2-octanol + ferrocyanide
octan-2-one + ferricyanide
-
enzyme electrode, 82% of vmax with 1-butanol, soluble enzyme, 80% of vmax with 1-butanol. With enzyme electrode, (S)-enantiomer is 82fold preferred over (R)-enantiomer, with soluble enzyme 800fold, respectively
-
-
?
(S)-glycidol + ferrocyanide
? + ferricyanide
-
enzyme electrode, 30% of vmax with 1-butanol, soluble enzyme, 1.6% of vmax with 1-butanol. With enzyme electrode, (S)-enantiomer is 0.9fold preferred over (R)-enantiomer, with soluble enzyme no disticnction
-
-
?
(S)-solketal + 2 ferricyanide
(S)-solketal aldehyde + 2 ferrocyanide + 2 H+
-
the second step in (S)-solketal conversion is much faster than the first one and that opposite enantiomeric preferences exist for the alcohol and the aldehyde substrates. (R)-enantiomer is prefered, enantiomeric ratio is 30 for initial rate measurement, 117 for progress curve analysis
-
-
r
(S)-solketal + ferrocyanide
? + ferricyanide
-
enzyme electrode, 9% of vmax with 1-butanol, 0.7% of vmax with 1-butanol. With enzyme electrode, (S)-enantiomer is 39fold preferred over (R)-enantiomer, with soluble enzyme 117fold, respectively
-
-
?
1,2-cyclohexanediol + ferricyanide
2-hydroxycyclohexanone + ferrocyanide
-
-
-
-
?
1,3-butanediol + ferricyanide
? + ferrocyanide
-
enzyme electrode, 91% of vmax with 1-butanol
-
-
?
1,3-cyclohexanediol + ferricyanide
3-hydroxycyclohexanone + ferrocyanide
-
-
-
-
?
1-butanol + N,N,N',N'-tetramethyl-p-phenylenediamine
butanal + ?
-
-
-
-
r
1-octanol + ferrocyanide
octanal + ferricyanide
-
enzyme electrode, 95% of vmax with 1-butanol, soluble enzyme, 95% of vmax with 1-butanol
-
-
?
1-octanol + N,N,N',N'-tetramethyl-p-phenylenediamine
octanal + ?
-
-
-
-
r
1-pentanol + ferrocyanide
pentanal + ferricyanide
-
enzyme electrode, 93% of vmax with 1-butanol, soluble enzyme, 100% of vmax with 1-butanol
-
-
?
1-pentanol + N,N,N',N'-tetramethyl-p-phenylenediamine
pentanal + ?
-
-
-
-
r
1-propanol + N,N,N',N'-tetramethyl-p-phenylenediamine
propanal + ?
-
-
-
-
r
2-butanol + ferrocyanide
butanone + ferricyanide
-
enzyme electrode, 17% of vmax with 1-butanol, soluble enzyme, 10% of vmax with 1-butanol
-
-
?
2-methylcyclohexanol + ferricyanide
2-methylcyclohexanone + ferrocyanide
-
-
-
-
?
2-octanol + ferrocyanide
octan-2-one + ferricyanide
-
enzyme electrode, 84% of vmax with 1-butanol, soluble enzyme, 80% of vmax with 1-butanol
-
-
?
2-pentanol + ferrocyanide
pentan-2-one + ferricyanide
-
enzyme electrode, 40% of vmax with 1-butanol, soluble enzyme, 34% of vmax with 1-butanol
-
-
?
2-propanol + ferrocyanide
acetone + ferricyanide
-
enzyme electrode, 9% of vmax with 1-butanol, soluble enzyme, 6% of vmax with 1-butanol
-
-
?
3-heptanol + ferrocyanide
heptan-3-one + ferricyanide
-
enzyme electrode, 3.7% of vmax with 1-butanol, soluble enzyme, 1% of vmax with 1-butanol
-
-
?
3-hexanol + ferrocyanide
hexan-3-one + ferricyanide
-
enzyme electrode, 6.5% of vmax with 1-butanol, soluble enzyme, 9% of vmax with 1-butanol
-
-
?
3-methylcyclohexanol + ferricyanide
3-methylcyclohexanone + ferrocyanide
-
-
-
-
?
3-octanol + ferrocyanide
octan-3-one + ferricyanide
-
enzyme electrode, 10% of vmax with 1-butanol, soluble enzyme, 10% of vmax with 1-butanol
-
-
?
3-pentanol + ferrocyanide
pentan-3-one + ferricyanide
-
enzyme electrode, 1% of vmax with 1-butanol, soluble enzyme, 1.4% of vmax with 1-butanol
-
-
?
4-methyl-2-pentanol + ferricyanide
4-methylpentan-2-one + ferrocyanide
-
enzyme electrode, 5.6% of vmax with 1-butanol
-
-
?
5-methyl-2-hexanol + ferricyanide
5-methylhexan-2-one + ferrocyanide
-
-
-
-
r
6-aminohexan-1-ol + N,N,N',N'-tetramethyl-p-phenylenediamine
6-aminohexanal + ?
-
-
-
-
r
acetaldehyde + reduced N,N,N',N'-tetramethyl-p-phenylenediamine
ethanol + N,N,N',N'-tetramethyl-p-phenylenediamine
-
-
-
-
r
benzyl alcohol + N,N,N',N'-tetramethyl-p-phenylenediamine
benzaldehyde + ?
-
-
-
-
r
butanal + reduced N,N,N',N'-tetramethyl-p-phenylenediamine
1-butanol + N,N,N',N'-tetramethyl-p-phenylenediamine
-
-
-
-
r
butane-1,3-diol + N,N,N',N'-tetramethyl-p-phenylenediamine
?
-
-
-
-
r
cyclohexanol + ferricyanide
cyclohexanone + ferrocyanide
-
enzyme electrode, 11% of vmax with 1-butanol, soluble enzyme, 9% of vmax with 1-butanol
-
-
?
ethanol + cytochrome c
ethanal + reduced cytochrome c
-
5% of the activity with Wurster's Blue. In presence of phenazine methosulfate, 60% of the activity with Wurster's Blue
-
-
r
ethanol + N,N,N',N'-tetramethyl-p-phenylenediamine
ethanal + ?
-
i.e. Wurster's Blue
-
-
?
ethanol + oxidized 2,6-dichlorophenolindophenol
ethanal + reduced 2,6-dichlorophenolindophenol
-
10% of the activity with Wurster's Blue. In presence of phenazine methosulfate, 75% of the activity with Wurster's Blue
-
-
r
formaldehyde + N,N,N',N'-tetramethyl-p-phenylenediamine
methanol + ?
-
-
-
-
r
octanal + reduced N,N,N',N'-tetramethyl-p-phenylenediamine
1-octanol + N,N,N',N'-tetramethyl-p-phenylenediamine
-
-
-
-
r
rac-2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane + ferricyanide
? + ferrocyanide
-
i.e. solketal. Enantiomeric ratio is 30 for solketal and 6 for rac-2,2-dimethyl-4-[1,1-2H]hydroxymethyl-1,3-[5,5,4-2H]dioxolane, d5-solketal. Isotopic substitution affects the relative kinetic weights of the initial hydron/deuteron transfer from substrate to cofactor and the subsequent proton/deuteron shift in the cofactor-product complex
-
-
?
(R)-solketal aldehyde + ferrocyanide
(R)-solketal + ferricyanide
-
-
-
-
r
(R)-solketal aldehyde + ferrocyanide
(R)-solketal + ferricyanide
-
(R)-enantiomer is prefered, enantiomeric ratio is 2.8 for initial rate measurement
-
-
r
1-butanol + ferricyanide
butanal + ferrocyanide
-
maximum velocity rate for both enzyme electrode and soluble enzyme
-
-
?
1-propanol + ferricyanide
propanal + ferrocyanide
-
enzyme electrode, 93% of vmax with 1-butanol, soluble enzyme, 90% of vmax with 1-butanol
-
-
?
3-methyl-2-pentanol + ferricyanide
3-methylpentan-2-one + ferrocyanide
-
enzyme electrode, 3.7% of vmax with 1-butanol
-
-
?
3-methyl-2-pentanol + ferricyanide
3-methylpentan-2-one + ferrocyanide
-
enzyme electrode, 9.3% of vmax with 1-butanol
-
-
?
3-nonanol + ferricyanide
nonan-3-one + ferrocyanide
-
enzyme electrode, 4.7% of vmax with 1-butanol, soluble enzyme, 4.5% of vmax with 1-butanol
-
-
?
4-decanol + ferricyanide
decan-4-one + ferrocyanide
-
enzyme electrode, 3.7% of vmax with 1-butanol, soluble enzyme, 3.6% of vmax with 1-butanol
-
-
?
4-heptanol + ferricyanide
heptan-4-one + ferrocyanide
-
enzyme electrode, 9.3% of vmax with 1-butanol, soluble enzyme, 10% of vmax with 1-butanol
-
-
?
ethanol + ferricyanide
ethanal + ferrocyanide
-
100% of the activity with Wurster's Blue
-
-
r
ethanol + ferricyanide
ethanal + ferrocyanide
-
86% of vmax with 1-butanol, soluble enzyme, 75% of vmax with 1-butanol
-
-
?
additional information
?
-
-
no substrate: D-glucose, D-galactose, D-fructose, Alpha-D-methylglucoside, mannitol
-
-
?
additional information
?
-
-
The affinity of the enzyme for aliphatic alcohols increases with the chain length of the substrate. The same property is observed for secondary alcohols in the series 2-propanol to 2-octanol
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
heme
-
binding of pyrroloquinoline quinone induces shifts in the resonances of the methyl groups of the heme porphyrin ring in the oxidized form of the apoenzyme and a shift in the methionine heme ligand resonance of the reduced form of the apoenzyme. A major effect of pyrroloquinoline quinone binding to apo-QH-EDH is a rotation of the methionine ligand of heme c. Pyrroloquinoline quinone becomes tightly bound, the event leading to a compact enzyme conformation which is able to catalyze rapid intramolecular electron transfer
heme
-
holoenzyme contains equimolar amounts of pyrroloquinoline quinone, Ca2+ and covalently bound heme. Low-spin heme protein
heme
-
in the crystals, the four hemes in the unit cell have only two different orientations, related by an 180° rotation about the b axis. The heme rings are oriented parallel to the b axis
pyrroloquinoline quinone
-
binding of pyrroloquinoline quinone induces shifts in the resonances of the methyl groups of the heme porphyrin ring in the oxidized form of the apoenzyme and a shift in the methionine heme ligand resonance of the reduced form of the apoenzyme. A major effect of pyrroloquinoline quinone binding to apo-QH-EDH is a rotation of the methionine ligand of heme c. Pyrroloquinoline quinone becomes tightly bound, the event leading to a compact enzyme conformation which is able to catalyze rapid intramolecular electron transfer
pyrroloquinoline quinone
-
holoenzyme contains equimolar amounts of pyrroloquinoline quinone, Ca2+ and covalently bound heme. Reconstitution of apoenzyme with pyrroloquinoline quinone analogues results in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Possession of the o-quinone or o-quinol moiety is not essential for binding but it is for activity
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
Ca2+
-
holoenzyme contains equimolar amounts of pyrroloquinoline quinone, Ca2+ and covalently bound heme
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.75
4-methyl-2-pentanol
-
enzyme electrode, pH 7.7, presence of CaCl2
0.24
5-methyl-2-hexanol
-
enzyme electrode, pH 7.7, presence of CaCl2
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
to 1.44 A resolution. The N-terminal domain has a beta-propeller fold and binds one pyrroloquinoline quinone cofactor and one calcium ion in the active site. A tetrahydrofuran-2-carboxylic acid molecule is present in the substrate-binding cleft. The C-terminal domain is an -helical type I cytochrome c with His608 and Met647 as heme-iron ligands. An unusual disulfide bond between two adjacent cysteines bridges the redox centers. It appears essential for electron transfer. A water channel delineates a possible pathway for proton transfer from the active site to the solvent
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, 0.15 M potassium phosphate buffer, pH 7.0, storage for routine purposes
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
biotechnology
-
co-immobilization of enzyme with redox polymer poly(vinylpyridine) complex functionalized with osmium bis(bipyridine) chloride on an electrode. The enzyme electrode readily oxidizes primary alcohols and secondary alcohols with maximum current densities varying between 0.43 and 0.98 A per m2 depending on the substrate and the operation temperature. The enzyme electrode is enantioselective in the oxidation of secondary alcohols. A strong preference is observed for the (S)-2-alcohols, the enantioselectivity increases with increasing chain length. The enantiomeric ratio E increases from 13 for (R,S)-2-butanol to approximately 80 for (R,S)-2-heptanol and (R,S)-2-octanol
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Groen, B.W.; van Kleef, M.A.G.; Duine, J.A.
Quinohaemoprotein alcohol dehydrogenase apoenzyme from Pseudomonas testosteroni
Biochem. J.
234
611-615
1986
Comamonas testosteroni
De Jong, G.A.H.; Caldeira, J.; Sun, J.; Jongejan, J.A.; de Vries, S.; Loehr, T.M.; Moura, I.; Moura, J.J.G.; Duine, J.A.
Characterization of the interaction between PQQ and heme c in the quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni
Biochemistry
34
9451-9458
1995
Comamonas testosteroni
De Jong, G.A.H.; Geerlof, A.; Stoorvogel, J.; Jongejan, J.A.; de Vries, S.; Duine, J.a.
Quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni. Purification, characterization, and reconstitution of the apoenzyme with pyrolloquinoline quinone analogues
Eur. J. Biochem.
230
899-905
1995
Comamonas testosteroni
Jongejan, A.; Jongejan, J.A.; Hagen, W.R.
Deuterium isotope effect on enantioselectivity in the Comamonas testosteroni quinohemoprotein alcohol dehydrogenase-catalyzed kinetic resolution of rac-2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane, solketal
Biochim. Biophys. Acta
1647
297-302
2003
Comamonas testosteroni
Oubrie, A.; Rozeboom, H.J.; Kalk, K.H.; Huizinga, E.G.; Dijkstra, B.W.
Crystal structure of quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni: structural basis for substrate oxidation and electron transfer
J. Biol. Chem.
277
3727-3732
2002
Comamonas testosteroni (Q46444)
Oubrie, A.; Huizinga, E.G.; Rozeboom, H.e.J.; Kalk, K.H.; de Jong, G.A.H.; Duine, J.A.; Dijkstra, B.W.
Crystallization of quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni: crystals with unique optical properties
Acta Crystallogr. Sect. D
D57
1732-1734
2001
Comamonas testosteroni
Somers, W.A.C.; Stigter, E.C.A.; Van Hartingsveldt, W.; Van Der Lugt, J.P.
Enantioselective oxidation of secondary alcohols at a quinohemoprotein alcohol dehydrogenase electrode
Appl. Biochem. Biotechnol.
75
151-161
1999
Comamonas testosteroni
-
Geerlof, A.; Rakels, J.J.L.; Straathof, A.J.J.; Heijnen, J.J.; Jongejan, J.A.; Duine, J.A.
Description of the kinetic mechanism and the enantioselectivity of quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni in the oxidation of alcohols and aldehydes
Eur. J. Biochem.
226
537-546
1994
Comamonas testosteroni
Stigter, E.C.A.; van der Lugt, J.P.; Somers, W.A.C.
Enantioselective oxidation of secondary alcohols by quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni
J. Mol. Catal. B
2
291-297
1997
Comamonas testosteroni
-
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