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Information on EC 4.2.1.28 - propanediol dehydratase and Organism(s) Klebsiella oxytoca and UniProt Accession Q59471
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4.2.1.28 propanediol dehydrataseIUBMB Comments
Two different forms of the enzyme have been described. One form requires a cobamide coenzyme, while the other is a glycyl radical enzyme. The cobamide-dependent enzyme has been shown to also dehydrate ethylene glycol to acetaldehyde.
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Word Map
- 4.2.1.28
-
cobiialamin
- oxytoca
- cyanocobalamin
- 5'-deoxyadenosine
-
homolysis
- propionaldehyde
- 1,2-ethanediol
-
hydroxocobalamine
- cobamide
- 5,6-dimethylbenzimidazole
-
cobalt-carbon
-
microcompartment
-
spectator
-
base-on
-
carbon-cobalt
-
corrins
-
5\'-deoxyadenosyl
- acetobacterium
- synthesis
- degradation
The taxonomic range for the selected organisms is: Klebsiella oxytoca
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Synonyms
diol dehydratase, diol dehydrase, propanediol dehydratase, pducde, adenosylcobalamin-dependent diol dehydratase, dioldehydrase, dioldehydratase, gldcde, adocbl-dependent diol dehydratase, 1,2-propanediol dehydratase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
1,2-propanediol dehydratase
-
-
-
-
1,2-propanediol hydro-lyase
adenosylcobalamin-dependent diol dehydratase
coenzyme B12-dependent diol dehydrase
-
-
-
-
dehydratase, diol
-
-
-
-
dehydratase, propanediol
-
-
-
-
diol dehydrase
-
-
-
-
diol dehydratase
dioldehydrase
-
-
-
-
dioldehydratase
-
-
-
-
DL-1,2-propanediol hydro-lyase
meso-2,3-butanediol dehydrase
-
-
-
-
Propanediol dehydrase
-
-
-
-
1,2-propanediol hydro-lyase
-
-
-
-
adenosylcobalamin-dependent diol dehydratase
-
-
-
-
diol dehydratase
-
-
-
-
diol dehydratase
-
-
DL-1,2-propanediol hydro-lyase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
propane-1,2-diol = propanal + H2O
binding of the substrate to the active site converts a hexacoordinated complex of K+ into the heptacoordinated one. A relatively large binding energy is released upon coordination of the two hydroxyl groups to K+, displacing a sixth ligand, H2O. Interaction of the coenzyme with the enzyme cleaves its Co-C bond, forming an adenosyl radical and cob(II)alamin. The radical abstracts the pro-S hydrogen of the S-enantiomer forming a substrate-derived radical and 5'-deoxyadenosine. The substrate-radical undergoes the 1,2-shift of the hydroxyl group, forming a product-derived gem-diol radical. The C2 of the product radical, abstracts a hydrogen back from deoxyadenosine with inversion of the configuration of C2, producing a 1,1-gem-diol, which undergoes dehydration, forming propionaldehyde and H2O
-
propane-1,2-diol = propanal + H2O
enzymatic radical catalysis, with adenosylcobalamin, coenzyme B12, as a cofactor. Dehydration of 1,2-diols to the corresponding aldehydes. The substrate and an essential potassium ion are located inside a betaalpha8 barrel. Two hydroxyl groups of the substrate coordinate directly to the potassium ion which binds to the negatively charged inner part of the cavity. Cobalamin bound covers the cavity to isolate the active site from the solvent. The initial migration of the hydroxyl group is stereospecific and the dehydration of a gem-diol undergoes steric control by the enzyme
-
propane-1,2-diol = propanal + H2O
the enzyme-bound adenosylcobalamin serves as an intermediate hydrogen carrier, accepting a hydrogen atom from C1 of the substrate to C5' of the coenzyme and giving a hydrogen back to C2 of the product. R and S enantiomers are bound to the active site of the enzyme in a symmetrical mode with respect to the plane
-
propane-1,2-diol = propanal + H2O
minimal mechanism for AdoCbl-dependent diol dehydratase involving the cob(II)alamin cofactor and active-site structure of the enzyme, overview
-
propane-1,2-diol = propanal + H2O
reaction mechanism, quantum mechanical/molecular mechanical, QM/MM, modeling of diol dehydratase based on the crystal structure of diol dehydratase-adeninylpentylcobalamin complex, overview. The hydrogen recombination is the rate-determining step for the overall reaction
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
elimination
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
propane-1,2-diol hydro-lyase (propanal-forming)
Two different forms of the enzyme have been described. One form requires a cobamide coenzyme, while the other is a glycyl radical enzyme. The cobamide-dependent enzyme has been shown to also dehydrate ethylene glycol to acetaldehyde.
CAS REGISTRY NUMBER
COMMENTARY
9026-90-8
-
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
1,2-propanediol
propanal
-
the substrate is located near to K+ rather than to the cobalt atom of cobalamin
-
?
propane-1,2-diol
propanal + H2O
-
Co-C bond formation during reaction
-
-
?
propane-1,2-diol
propanal + H2O
-
substrate binding structure and mechanism, overview
-
-
?
additional information
?
-
-
the enzyme catalyze the conversion of 1,2-diols, such as 1,2-propanediol, ethanediol and glycerol, to the corresponding aldehydes
-
-
?
additional information
?
-
-
hydrogen bonding interaction between the hydroxyl group on C2 of substrate and the side chain of residue Hisalpha143 is important not only for catalysis but also for protecting radical intermediates, overview
-
-
?
additional information
?
-
-
Asp335 has a strong anticatalytic effect on the OH group migration despite its important role in substrate binding. The synergistic interplay of the O-C bond cleavage by Ca2+ ion and the deprotonation of the spectator OH-group by Glu170 is required to overcome the anticatalytic effect of Asp335
-
-
?
NATURAL SUBSTRATE
NATURAL 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
additional information
?
-
-
the enzyme catalyze the conversion of 1,2-diols, such as 1,2-propanediol, ethanediol and glycerol, to the corresponding aldehydes
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Cobalamin
-
roles of adenine anchoring and ion pairing at the coenzyme B12-binding site, overview. Presence of a positive charge at the beta135 residue increases the affinity for cobalamins but is not essential for catalysis, and the introduction of a negative charge there prevents the enzyme-cobalamin interaction
[4Fe-4S]-center
presence of 2.80 equivalents of iron and 2.71 ± 0.02 equivalents of sulfur per monomer
coenzyme B12
-
adenosylcobalamin. Glycerol-inactivated and oxygen inactivated enzyme undergoes rapid reactivation in the presence of the cofactor, ATP and Mg2+
coenzyme B12
-
adenosylcobalamin. Glycerol-inactivated and oxygen inactivated enzyme undergoes rapid reactivation in the presence of the reactivating factor, the cofactor, ATP and Mg2+
coenzyme B12
-
adenosylcobalamin. Glycerol-inactivated enzyme undergoes reactivation in the presence of the reactivating factor, the cofactor, ATP and Mg2+
coenzyme B12
-
adenosylcobalamin. There is a strict specificity of the enzyme for the coenzyme adenosyl group
coenzyme B12
-
adenosylcobalamin, dependent on, two mol of 5'-deoxyadenosine per mol of enzyme are formed as an inactivation product from the coenzyme adenosyl group in presence of inhibitors, overview
additional information
-
adeninylpentylcobalamin and cyanocobalamin are inactive as cofactors
-
additional information
-
adenosylcobinamide 3-benzimidazolylpropyl phosphate, beta-adenosyl-alpha-benzimidazolylcobamide and beta-adenosyl-alpha-imidazolylcobamide are active coenzymes. No coenzyme activity: adenosylcobinamide 3-(2-methylbenzimidazolyl)propyl phosphate
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
Co2+
K+
Mg2+
additional information
Ca2+
-
1.6-1.9 mol/mol of enzyme. CaCl2 and substrate show not only a stabilizing effect but also some activating effect of about 30%
Ca2+
-
directly coordinates to substrate and is essential for structural retention and substrate binding
Co2+
-
in the cobalamin cofactor, presence of a positive charge at the beta135 residue increases the affinity for cobalamins but is not essential for catalysis, and the introduction of a negative charge there prevents the enzyme-cobalamin interaction
additional information
-
no requirements of divalentmetal ions for catalysis, metal contents in purified recombinant enzyme
additional information
-
the activation energy for the OH group migration, which is essential in the conversion of diols to corresponding aldehydes, is sensitive to the identity of the metal ion
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
cyanocobalamin
a tightly bound inactive coenzyme analogue lacking the adenine ring in the upper axial ligand, imitates the inactivated cofactor
glycerol
adenosylcobalamin-dependent diol dehydratase undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co-C bond. The damaged cofactor remains tightly bound to the active site, it is not displaced by intact adenosylcobalmin, resulting in the irreversible inactivation of the enzyme. The DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg2+ by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme, overview
3-butene-1,2-diol
-
holodiol dehydratase undergoes rapid and irreversible inactivation, the inactivation cleaves the Co-C bond of adenosylcobalamin irreversibly forming unidentified radicals and cob(II)alamin that resist oxidation even in the presence of oxygen, inactivation mechanism, overview
3-butyne-1,2-diol
-
holodiol dehydratase undergoes rapid and irreversible inactivation, the inactivation cleaves the Co-C bond of adenosylcobalamin irreversibly forming unidentified radicals and cob(II)alamin that resist oxidation even in the presence of oxygen, inactivation mechanism, overview
adenosylcobinamide 3-(2-methylbenzimidazolyl)propyl phosphate
-
competitive inhibitor with respect to coenzyme B12. Irreversible cleavage of the coenzyme Co-C bond during the inactivation
-
cyanocobalamin
a tightly bound inactive coenzyme analogue lacking the adenine ring in the upper axial ligand, imitates the inactivated cofactor
EDTA
-
EDTA inhibition of apoenzyme is reversible at least in the initial phase
thioglycerol
-
holodiol dehydratase undergoes rapid and irreversible inactivation, the inactivation cleaves the Co-C bond of adenosylcobalamin irreversibly forming unidentified radicals and cob(II)alamin that resist oxidation even in the presence of oxygen, inactivation mechanism, overview
adenosylmethylcobalamin
-
catalytic efficiency (turnover number to Km-value) of the holoenzyme with adenosylmethylcobalamin is 0.15% of that for the regular coenzyme adenosylcobalamin, Km: 0.0017 mM
glycerol
adenosylcobalamin-dependent diol dehydratase undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co-C bond. The damaged cofactor remains tightly bound to the active site, it is not displaced by intact adenosylcobalamin, resulting in the irreversible inactivation of the enzyme. The DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg2+ by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme, overview
glycerol
adenosylcobalamin-dependent diol dehydratase undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co-C bond. The damaged cofactor remains tightly bound to the active site, it is not displaced by intact Ado-Cbl, resulting in the irreversible inactivation of the enzyme. The DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg2+ by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme, overview
Propane-1,2-diol
-
leads to inactivation of wild-type and mutant enzymes during catalysis, kinetics, overview
Propane-1,2-diol
-
mechanism-based inactivation obeying first-order reaction kinetics
additional information
-
inactivated holoenzymes undergo reactivation by diol dehydratase-reactivating factor in the presence of ATP, Mg2+ and adenosylcobalamin
-
additional information
-
Asp335 has a strong anticatalytic effect on the OH group migration despite its important role in substrate binding. The synergistic interplay of the O-C bond cleavage by Ca2+ ion and the deprotonation of the spectator OH-group by Glu170 is required to overcome the anticatalytic effect of Asp335
-
additional information
-
no inhibition by 5 mM of 1,10-phenanthroline, 2,2'-dipyridyl, citrate, succinate, tartrate, malate, and salicylate
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
diol dehydratase-reactivating factor
both dehydratase-reactivating factors exist as alpha2beta2 heterotetramers, DdrA or GdrA and DdrB or GdrB, respectively. The diol dehydratase DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg2+ by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme, overview. Mechanism of the reactivation of inactivated holoenzymes by reactivating factors, overview. The enzyme does not form a complex with the reactivating factor while it exists as an active holoenzyme. The reactivating factor binds ATP and hydrolyzes it to ADP by its own weak ATPase activity. The resulting ADP-bound form of the reactivating factor has a high affinity for the enzyme, and interacts with the inactivated holoenzyme to form a tight apoenzyme/reactivating factor complex, with the concomitant release of the damaged cofactor. The reactivating factor reverts to a low-affinity form through the replacement of bound ADP by free ATP, resulting in the dissociation of the apoenzyme/reactivating factor complex into apoenzyme and the reactivating factor, kinetics, overview
-
additional information
-
ADP and ATP analogues are not able to substitute ATP significantly
-
ATP
-
absolutely required for the reactivation of the inactivated holoenzyme by the reactivating factor
diol dehydratase-reactivating factor
-
activates inactive enzymeCN-Cbl complexes, dimeric (alphabbeta) structurea large subunit of 64 kDa and a small subunit of 14 kDa
-
diol dehydratase-reactivating factor
both dehydratase-reactivating factors exist as alpha2beta2 heterotetramers, DdrA or GdrA and DdrB or GdrB, respectively. The diol dehydratase DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg2+ by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme, overview. Mechanism of the reactivation of inactivated holoenzymes by reactivating factors, overview. The enzyme does not form a complex with the reactivating factor while it exists as an active holoenzyme. The reactivating factor binds ATP and hydrolyzes it to ADP by its own weak ATPase activity. The resulting ADP-bound form of the reactivating factor has a high affinity for the enzyme, and interacts with the inactivated holoenzyme to form a tight apoenzyme/reactivating factor complex, with the concomitant release of the damaged cofactor. The reactivating factor reverts to a low-affinity form through the replacement of bound ADP by free ATP, resulting in the dissociation of the apoenzyme/reactivating factor complex into apoenzyme and the reactivating factor, kinetics, overview
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3.9
(R)-Propane-1,2-diol
mutant enzyme S301A/Q336A, at pH 8.0 and 37°C
2.6
(S)-propane-1,2-diol
mutant enzyme S301A/Q336A, at pH 8.0 and 37°C
0.15
Propan-1,2-diol
-
pH 8.0, 37°C, wild-type enzyme and mutant Salpha224A
0.39
Propan-1,2-diol
-
pH 8.0, 37°C, mutant Kbeta135A and mutant Kbeta135Q
0.031
Propane-1,2-diol
mutant T172A, pH not specified in the publication, temperature not specified in the publication
0.15
Propane-1,2-diol
wild-type, pH not specified in the publication, temperature not specified in the publication
0.15
Propane-1,2-diol
mutant S224A, pH not specified in the publication, temperature not specified in the publication
0.16
Propane-1,2-diol
mutant T172S, pH not specified in the publication, temperature not specified in the publication
0.51
Propane-1,2-diol
mutant S224C, pH not specified in the publication, temperature not specified in the publication
1.9
Propane-1,2-diol
mutant S224N, pH not specified in the publication, temperature not specified in the publication
additional information
additional information
-
kinetic analysis of mutant enzymes, overview
-
additional information
additional information
-
kinetic analysis with substrate analogues 3-butene-1,2-diol, 3-butyne-1,2-diol or thioglycerol
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.5
Propane-1,2-diol
mutant T172A, pH not specified in the publication, temperature not specified in the publication
12
Propane-1,2-diol
mutant S224C, pH not specified in the publication, temperature not specified in the publication
17
Propane-1,2-diol
mutant S224N, pH not specified in the publication, temperature not specified in the publication
34
Propane-1,2-diol
mutant T172S, pH not specified in the publication, temperature not specified in the publication
64
Propane-1,2-diol
mutant S224A, pH not specified in the publication, temperature not specified in the publication
336
Propane-1,2-diol
wild-type, pH not specified in the publication, temperature not specified in the publication
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
9
Propane-1,2-diol
mutant S224N, pH not specified in the publication, temperature not specified in the publication
16
Propane-1,2-diol
mutant T172A, pH not specified in the publication, temperature not specified in the publication
24
Propane-1,2-diol
mutant S224C, pH not specified in the publication, temperature not specified in the publication
210
Propane-1,2-diol
mutant T172S, pH not specified in the publication, temperature not specified in the publication
430
Propane-1,2-diol
mutant S224A, pH not specified in the publication, temperature not specified in the publication
2200
Propane-1,2-diol
wild-type, pH not specified in the publication, temperature not specified in the publication
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0027
adenosylcobinamide 3-(2-methylbenzimidazolyl)propyl phosphate
-
-
-
additional information
additional information
-
kinetic analysis with inhibitory substrate analogues 3-butene-1,2-diol, 3-butyne-1,2-diol or thioglycerol
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
-
ATCC8724 cells grown without aeration in glycerol-1,2-propanediol medium
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
physiological function
enzyme undergoes mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co-C bond. Diol dehydratase activity is maintained through coenzyme recycling by a reactivating system for diol dehydratase composed of diol dehydratase-reactivase, PduO adenosyltransferase, and a reducing system. The releasing factor diol dehydratase-reactivase is essential for the recycling of adenosycobalamin, and PduO is a specific adenosylating enzyme for the diol dehydratase reactivation, whereas CobA and EutT exert their effects through free synthesized coenzyme
physiological function
mechanistic comparison between glycyl radical-dependent enzyme from Roseburia inulinivorans and vitamin B12-dependent enzyme from Klebsiella oxytoca. Unlike B12-dependent propanediol dehydratase, the glycyl radical-dependent enzyme appears to catalyze the direct elimination of a hydroxyl group from an initially formed substrate-based radical, avoiding the generation of a 1,1-gem diol intermediate
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). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hexamer
additional information
-
dimer of a heterotrimer, alphabetagamma2. The enzyme dissociates into two dissimilar protein components, F and S, upon DEAE-cellulose chromatography in the absence of substrate, which are identified as the monomeric beta subunit and the trimeric alpha2gamma2 complex, respectively
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
as complexes with coenzyme analogs in the substrate-bound and substrate-free forms
-
complexed with cyanocobalamin and glycerol, sandwich-drop vapor diffusion method, using 20 mM Tris/HCl buffer (pH 8.0) containing 14%-18% (w/v) poly(ethylene glycol) 6000, 0.23-0.25 M ammonium sulfate and 0.2% lauryl dimethylamine oxide
structures in the absence and presence of substrates at resolutions of 1.55-2.05 A
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D335A
E170A E170Q
computational mutation study. The spectator OH group is not fully activated in the Glu170Gln and Glu170Ala mutants during the OH group migration, and thus the activation energies in the Glu170Gln and Glu170Ala mutants are higher than that in the wild-type enzyme
E170Q
computational mutation study. The spectator OH group is not fully activated in the Glu170Gln and Glu170Ala mutants during the OH group migration, and thus the activation energies in the Glu170Gln and Glu170Ala mutants are higher than that in the wild-type enzyme
H143A
Halpha143A
-
site-directed mutagenesis, the mutant shows residual activity compared to the wild-type enzyme
Halpha143E
-
site-directed mutagenesis, the mutant is inactive and does not form (alphabeta)2 complexes
Halpha143K
-
site-directed mutagenesis, the mutant is inactive and does not form (alphabeta)2 complexes
Halpha143L
-
site-directed mutagenesis, the mutant shows residual activity compared to the wild-type enzyme
Halpha143Q
-
site-directed mutagenesis, the mutant shows residual activity compared to the wild-type enzyme, irreversible inactivation by O2 in the absence of substrate at a much lower rate than the wild type, preference of the Halpha143Q mutant for (R)- and (S)-1,2-propanediols, kinetic parameters for each enantiomer, overview
Kbeta135A
-
site-directed mutagenesis, the mutant shows 42% reduced activity compared to the wild-type enzyme, the mutant is less sensitive to inhibitor CN-cobalamin
Kbeta135E
-
site-directed mutagenesis, the mutant shows 98% reduced activity compared to the wild-type enzyme
Kbeta135Q
-
site-directed mutagenesis, the mutant shows 27% reduced activity compared to the wild-type enzyme, the mutant is less sensitive to inhibitor CN-cobalamin
Kbeta135R
-
site-directed mutagenesis, the mutant shows 24% reduced activity compared to the wild-type enzyme
Q336A
the mutant shows decreased activity (21%) compared to the wild type enzyme
S224A
mutation in alpha-subunit, a key residue for stabilizing the post-homolysis state of the adenosyl group. More than 5fold reduction in kcat/Km
S224C
mutation in alpha-subunit, a key residue for stabilizing the post-homolysis state of the adenosyl group. About 100fold reduction in kcat/Km
S224N
mutation in alpha-subunit, a key residue for stabilizing the post-homolysis state of the adenosyl group. More than 100fold reduction in kcat/Km
S301A
the mutant shows decreased activity (76%) compared to the wild type enzyme
S301A/Q336A
the mutant shows lowest activity (9.8%) compared to the wild type enzyme
Salpha224A
-
site-directed mutagenesis, the mutant shows 81% reduced activity compared to the wild-type enzyme, mechanism-based complete inactivation of the Salpha224A holoenzyme during catalysis by propan-1,2-diol leading to accumulation of cobalamin, mechanism, overview
Salpha224N
-
site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme
T172A
mutation in alpha-subunit, a key residue for stabilizing the post-homolysis state of the adenosyl group. More than 100fold reduction in kcat/Km
T172S
mutation in alpha-subunit, a key residue for stabilizing the post-homolysis state of the adenosyl group. More than 10fold reduction in kcat/Km
additional information
-
construction of chimeric enzymes with subunit compositions alphaGbetaD2GgammaG and alphaGbetaGgammaDG, overview
D335A
computational mutation study. The OH group migration is accelerated in the Asp335Ala mutant, due to the absence of the electric repulsion between Asp335 and the migrating OH group
H143A
computational mutation study. The resonance stabilization of the transition state in the OH group migration is observed in the wild-type enzyme while not in the His143Ala mutant. Since the cleavage of the C2-oxygen bond of 1,2-diol radical proceeds in a more homolytic manner in the His143Ala mutant, Glu170 cannot effectively deprotonate the spectator OH group in the transition state, leading to increased activation energy of the OH group migration in the His143Ala mutant
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
calcium-deprived apoenzyme is unstable when incubated at 37°C in the absence of both Ca2+ and substrate
-
limited proteolysis of the enzyme with trypsin converts the enzyme into a highly soluble form without loss of activity
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
beta-subunit DNA and amino acid sequence determination and analysis, sequence comparison, and expression of wild-type and chimeric mutants, in Escherichia coli strain BL21(DE3), distribution of the chimeric mutants in recombinant bacterial cells, overview
-
expression in Escherichia coli
N-terminal truncations (20 or 16 amino acid residues) of either or both of the beta and gamma subunits are expressed on a high level in Escherichia coli. All the mutant enzymes obtained are expressed in a soluble, active form. The mutant enzyme with the N-terminal truncations of both beta and gamma subunits are indistinguishable in catalytic properties from recombinant wild-type enzyme of the enzyme purified from Klebsiella oxytoca in a soluble form
-
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
inactivated holoenzymes undergo reactivation by diol dehydratase-reactivating factor in the presence of ATP, Mg2+ and adenosylcobalamin
-
reconstitution of calcium-containing apoenzyme from calcium-deprived apoenzyme and Ca2+, overview
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Tobimatsu, T.; Kajiura, H.; Toraya, T.
Specificities of reactivating factors for adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase
Arch. Microbiol.
174
81-88
2000
Klebsiella oxytoca, Klebsiella pneumoniae
Mori, K.; Toraya, T.
Mechanism of reactivation of coenzyme B12-dependent diol dehydratase by a molecular chaperone-like reactivating factor
Biochemistry
38
13170-13178
1999
Klebsiella oxytoca
Toraya, T.
Radical catalysis of B12 enzymes: structure, mechanism, inactivation, and reactivation of diol and glycerol dehydratases
Cell. Mol. Life Sci.
57
106-127
2000
Klebsiella oxytoca, Klebsiella pneumoniae
Fukuoka, M.; Yamada, S.; Miyoshi, S.; Yamashita, K.; Yamanishi, M.; Zou, X.; Brown, K.L.; Toraya, T.
Functions of the D-ribosyl moiety and the lower axial ligand of the nucleotide loop of coenzyme B(12) in diol dehydratase and ethanolamine ammonia-lyase reactions
J. Biochem.
132
935-943
2002
Klebsiella oxytoca
Shibata, N.; Nakanishi, Y.; Fukuoka, M.; Yamanishi, M.; Yasuoka, N.; Toraya, T.
Structural rationalization for the lack of stereospecificity in coenzyme B12-dependent diol dehydratase
J. Biol. Chem.
278
22717-22725
2003
Klebsiella oxytoca
Masuda, J.; Shibata, N.; Morimoto, Y.; Toraya, T.; Yasuoka, N.
How a protein generates a catalytic radical from coenzyme B(12): X-ray structure of a diol-dehydratase-adeninylpentylcobalamin complex
Structure Fold. Des.
8
775-788
2000
Klebsiella oxytoca
Fukuoka, M.; Nakanishi, Y.; Hannak, R.B.; Krautler, B.; Toraya, T.
Homoadenosylcobalamins as probes for exploring the active sites of coenzyme B12-dependent diol dehydratase and ethanolamine ammonia-lyase
FEBS J.
272
4787-4796
2005
Klebsiella oxytoca
Tobimatsu, T.; Kawata, M.; Toraya, T.
The N-terminal regions of beta and gamma subunits lower the solubility of adenosylcobalamin-dependent diol dehydratase
Biosci. Biotechnol. Biochem.
69
455-462
2005
Klebsiella oxytoca
Kajiura, H.; Mori, K.; Shibata, N.; Toraya, T.
Molecular basis for specificities of reactivating factors for adenosylcobalamin-dependent diol and glycerol dehydratases
FEBS J.
274
5556-5566
2007
Klebsiella oxytoca, Klebsiella pneumoniae
Sakai, T.; Yamasaki, A.; Toyofuku, S.; Nishiki, T.; Yunoki, M.; Komoto, N.; Tobimatsu, T.; Toraya, T.
Construction and characterization of hybrid dehydratases between adenosylcobalamin-dependent diol and glycerol dehydratases
J. Nutr. Sci. Vitaminol.
53
102-108
2007
Klebsiella oxytoca, Klebsiella pneumoniae
Kawata, M.; Kinoshita, K.; Takahashi, S.; Ogura, K.; Komoto, N.; Yamanishi, M.; Tobimatsu, T.; Toraya, T.
Survey of Catalytic Residues and Essential Roles of Glutamate-aplpha170 and Aspartate-alpha335 in Coenzyme B12-dependent Diol Dehydratase
J. Biol. Chem.
281
18327-18334
2006
Klebsiella oxytoca
Tobimatsu, T.; Nishiki, T.; Morimoto, M.; Miyata, R.; Toraya, T.
Low-solubility glycerol dehydratase, a chimeric enzyme of coenzyme B(12)-dependent glycerol and diol dehydratases
Arch. Microbiol.
191
199-206
2008
Salmonella enterica, Klebsiella oxytoca, Klebsiella pneumoniae
Kinoshita, K.; Kawata, M.; Ogura, K.; Yamasaki, A.; Watanabe, T.; Komoto, N.; Hieda, N.; Yamanishi, M.; Tobimatsu, T.; Toraya, T.
Histidine-alpha143 assists 1,2-hydroxyl group migration and protects radical intermediates in coenzyme B12-dependent diol dehydratase
Biochemistry
47
3162-3173
2008
Klebsiella oxytoca
Ogura, K.; Kunita, S.; Mori, K.; Tobimatsu, T.; Toraya, T.
Roles of adenine anchoring and ion pairing at the coenzyme B12-binding site in diol dehydratase catalysis
FEBS J.
275
6204-6216
2008
Klebsiella oxytoca
Toraya, T.; Tamura, N.; Watanabe, T.; Yamanishi, M.; Hieda, N.; Mori, K.
Mechanism-based inactivation of coenzyme B12-dependent diol dehydratase by 3-unsaturated 1,2-diols and thioglycerol
J. Biochem.
144
437-446
2008
Klebsiella oxytoca
Toraya, T.; Honda, S.; Mori, K.
Coenzyme B12-dependent diol dehydratase is a potassium ion-requiring calcium metalloenzyme: evidence that the substrate-coordinated metal ion is calcium
Biochemistry
49
7210-7217
2010
Klebsiella oxytoca, Klebsiella oxytoca ATCC 8724
Mori, K.; Hosokawa, Y.; Yoshinaga, T.; Toraya, T.
Diol dehydratase-reactivating factor is a reactivase - evidence for multiple turnovers and subunit swapping with diol dehydratase
FEBS J.
277
4931-4943
2010
Klebsiella oxytoca (Q59470), Klebsiella oxytoca (Q59471), Klebsiella oxytoca (Q59472)
Kamachi, T.; Doitomi, K.; Takahata, M.; Toraya, T.; Yoshizawa, K.
Catalytic roles of the metal ion in the substrate-binding site of coenzyme B12-dependent diol dehydratase
Inorg. Chem.
50
2944-2952
2011
Klebsiella oxytoca
Yamanishi, M.; Kinoshita, K.; Fukuoka, M.; Saito, T.; Tanokuchi, A.; Ikeda, Y.; Obayashi, H.; Mori, K.; Shibata, N.; Tobimatsu, T.; Toraya, T.
Redesign of coenzyme B12 dependent diol dehydratase to be resistant to the mechanism-based inactivation by glycerol and act on longer chain 1,2-diols
FEBS J.
279
793-804
2012
Klebsiella oxytoca (Q59470), Klebsiella oxytoca ATCC 8724 (Q59470)
Shibata, N.; Sueyoshi, Y.; Higuchi, Y.; Toraya, T.
Direct participation of a peripheral side chain of a corrin ring in coenzyme B12 catalysis
Angew. Chem. Int. Ed. Engl.
57
7830-7835
2018
Klebsiella oxytoca (Q59470)
Toraya, T.; Tanokuchi, A.; Yamasaki, A.; Nakamura, T.; Ogura, K.; Tobimatsu, T.
Diol dehydratase-reactivase is essential for recycling of coenzyme B12 in diol dehydratase
Biochemistry
55
69-78
2016
Klebsiella oxytoca (Q59470 and Q59471 and Q59472), Klebsiella oxytoca, Klebsiella oxytoca ATCC 8724 (Q59470 and Q59471 and Q59472)
Levin, B.J.; Balskus, E.P.
Characterization of 1,2-propanediol dehydratases reveals distinct mechanisms for B12-dependent and glycyl radical enzymes
Biochemistry
57
3222-3226
2018
Roseburia inulinivorans, Klebsiella oxytoca (Q59470 and Q59471 and Q59472), Roseburia inulinivorans A2-194, Klebsiella oxytoca ATCC 8724 (Q59470 and Q59471 and Q59472)
Doitomi, K.; Kamachi, T.; Toraya, T.; Yoshizawa, K.
Computational mutation study of the roles of catalytic residues in coenzyme B12-dependent diol dehydratase
Bull. Chem. Soc. JPN
89
955-964
2016
Klebsiella oxytoca (Q59470)
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