Information on EC 4.1.3.43 - 4-hydroxy-2-oxohexanoate aldolase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.1.3.43
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RECOMMENDED NAME
GeneOntology No.
4-hydroxy-2-oxohexanoate aldolase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(S)-4-hydroxy-2-oxohexanoate = propanal + pyruvate
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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BphI exhibits a compulsory order mechanism, with pyruvate binding first
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
4-nitrotoluene degradation II
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androstenedione degradation
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Metabolic pathways
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Microbial metabolism in diverse environments
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Xylene degradation
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androgen and estrogen metabolism
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SYSTEMATIC NAME
IUBMB Comments
(S)-4-hydroxy-2-oxohexanoate pyruvate-lyase (propanal-forming)
Requires Mn2+ for maximal activity [1,2]. The enzymes from the bacteria Burkholderia xenovorans and Thermus thermophilus also perform the reaction of EC 4.1.3.39, 4-hydroxy-2-oxovalerate aldolase [1,2,6]. The enzyme forms a bifunctional complex with EC 1.2.1.87, propanal dehydrogenase (CoA-propanoylating), with a tight channel connecting the two subunits [3,4,6].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(4R)-4-hydroxy-2-oxohexanoate
pyruvate + propionaldehyde
show the reaction diagram
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reaction catalyzed by BphI variants Y290F, L87N/Y290F and L87W/Y290F
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?
(4R)-4-hydroxy-2-oxopentanoate
pyruvate + acetaldehyde
show the reaction diagram
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-
-
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?
(4S)-4-hydroxy-2-oxohexanoate
pyruvate + propionaldehyde
show the reaction diagram
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double variants L87N/Y290F and L87W/Y290F inactive toward (4S)-4-hydroxy-2-oxohexanoate
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?
(4S)-4-hydroxy-2-oxopentanoate
acetaldehyde + pyruvate
show the reaction diagram
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-
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?
(4S)-4-hydroxy-2-oxopentanoate
pyruvate + acetaldehyde
show the reaction diagram
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-
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?
(S)-4-hydroxy-2-oxohexanoate
propanal + pyruvate
show the reaction diagram
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-
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?
4-hydroxy-2-oxoheptanoate
pyruvate + butanal
show the reaction diagram
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-
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?
4-hydroxy-2-oxohexanoate
propanal + pyruvate
show the reaction diagram
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-
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?
4-hydroxy-2-oxohexanoate
propionaldehyde + pyruvate
show the reaction diagram
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4-hydroxy-2-oxohexanoate i.e. HOHA
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?
4-hydroxy-2-oxohexanoate
pyruvate + propionaldehyde
show the reaction diagram
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-
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?
4-hydroxy-2-oxopentanoate
acetaldehyde + pyruvate
show the reaction diagram
4-hydroxy-2-oxopentanoate
pyruvate + acetaldehyde
show the reaction diagram
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-
-
?
Oxaloacetate
CO2 + pyruvate
show the reaction diagram
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oxaloacetate decarboxylation is a secondary reaction of pyruvate aldolases
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?
Oxaloacetate
Pyruvate + CO2
show the reaction diagram
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?
pyruvate + acetaldehyde
(4R)-4-hydroxy-2-oxopentanoate
show the reaction diagram
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-
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?
pyruvate + acetaldehyde
(4S)-4-hydroxy-2-oxopentanoate
show the reaction diagram
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-
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?
pyruvate + acetaldehyde
(S)-4-hydroxy-2-oxopentanoate
show the reaction diagram
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?
pyruvate + butyraldehyde
?
show the reaction diagram
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?
pyruvate + glycolaldehyde
?
show the reaction diagram
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?
pyruvate + hexaldehyde
?
show the reaction diagram
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?
pyruvate + isobutyraldehyde
?
show the reaction diagram
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?
pyruvate + pentaldehyde
?
show the reaction diagram
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-
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?
pyruvate + propionaldehyde
(4R)-4-hydroxy-2-oxohexanoate
show the reaction diagram
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reaction catalyzed by BphI variants Y290F and Y290S
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?
pyruvate + propionaldehyde
(4S)-4-hydroxy-2-oxohexanoate
show the reaction diagram
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?
pyruvate + propionaldehyde
4-hydroxy-2-oxohexanoate
show the reaction diagram
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?
pyruvate + propionaldehyde
?
show the reaction diagram
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(S)-4-hydroxy-2-oxohexanoate
propanal + pyruvate
show the reaction diagram
P51015
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?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-oxobutanoate
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competitive inhibition
2-Oxopentanoate
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competitive inhibition
4-methyl-2-oxopentanoate
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competitive inhibition
Cd2+
56.0% activity at 0.1 mM and 5.4% activity at 1 mM chloride salt
Co2+
19.3% activity at 0.1 mM and 18.3% activity at 1 mM chloride salt
glyoxylate
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competitive inhibition
Mg2+
3.4% activity at 0.1 mM and 14.8% activity at 1 mM chloride salt
Mn2+
100% activity at 0.1 mM and 1 mM chloride salt
Ni2+
2.6% activity at 0.1 mM and 17.5% activity at 1 mM chloride salt
pyruvate
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competitive inhibition
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
BphJ
15fold increased activity
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.012 - 0.757
(4R)-4-hydroxy-2-oxopentanoate
0.013 - 0.228
(4S)-4-hydroxy-2-oxopentanoate
0.35
4-hydroxy-2-oxoheptanoate
pH 8.0, 25C
0.037 - 1.1
4-hydroxy-2-oxohexanoate
0.041 - 0.94
4-hydroxy-2-oxopentanoate
64.2 - 86.3
acetaldehyde
49.2 - 323
Butyraldehyde
124.6
glycolaldehyde
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app. Km-value with pyruvate as carbonyl donor, pH 8.0 and 25C
0.21 - 7.5
oxaloacetate
24.8 - 80.5
pentaldehyde
16.8 - 136
propionaldehyde
11 - 38.2
pyruvate
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.043 - 0.17
(4R)-4-hydroxy-2-oxopentanoate
0.089 - 4.07
(4S)-4-hydroxy-2-oxopentanoate
0.63
4-hydroxy-2-oxoheptanoate
Paraburkholderia xenovorans
P51015
pH 8.0, 25C
0.27 - 3.9
4-hydroxy-2-oxohexanoate
0.08 - 4.3
4-hydroxy-2-oxopentanoate
0.2 - 0.94
acetaldehyde
0.16 - 0.64
Butyraldehyde
0.4
glycolaldehyde
Paraburkholderia xenovorans
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app. kcat-value with pyruvate as carbonyl donor, pH 8.0 and 25C
0.05 - 16.5
oxaloacetate
0.13 - 0.58
pentaldehyde
0.1 - 1.79
propionaldehyde
0.11 - 1.2
pyruvate
additional information
additional information
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.1 - 14.1
(4R)-4-hydroxy-2-oxopentanoate
4449
0.012 - 44.7
(4S)-4-hydroxy-2-oxopentanoate
2805
1.8
4-hydroxy-2-oxoheptanoate
Paraburkholderia xenovorans
P51015
pH 8.0, 25C
28492
5.2 - 33.3
4-hydroxy-2-oxohexanoate
2062
0.8 - 46.1
4-hydroxy-2-oxopentanoate
1197
0.00042 - 0.0134
acetaldehyde
90
0.00016 - 0.0129
Butyraldehyde
499
0.0037
glycolaldehyde
Paraburkholderia xenovorans
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kcat(app)/Km(app) with pyruvate as carbonyl donor, pH 8.0 and 25C
604
0.00001 - 0.00013
hexaldehyde
6132
0.0005
Isobutyraldehyde
Paraburkholderia xenovorans
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kcat(app)/Km(app) with pyruvate as carbonyl donor, pH 8.0 and 25C
1646
0.0058 - 2.2
oxaloacetate
57
0.00004 - 0.0233
pentaldehyde
3401
0.0018 - 0.0175
propionaldehyde
273
0.0036 - 0.0915
pyruvate
31
additional information
additional information
2
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.34
2-oxobutanoate
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pH 8.0, 25C
1.09
2-Oxopentanoate
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pH 8.0, 25C
2.71
4-methyl-2-oxopentanoate
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pH 8.0, 25C
0.21
glyoxylate
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pH 8.0, 25C
0.00093
oxalate
pH 8.0, 25C
0.55 - 10.3
pyruvate
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32000
BphI-BphJ-complex, alpha2beta2, 2 * 32000 BphJ, 2 * 37000 BphI
72000
native molecular mass of TTHB246, determined by gel filtration
78000
native molecular mass of TTHB246, determined by static light scattering
140000
native molecular mass of the purified BphI-BphJ-complex, estimated by gel filtration
142000
native molecular mass of TTHB246-TTHB247 complex, determined by gel filtration
144000
molecular mass of TTHB246-BphJ chimeric complex, determined by gel filtration
145000
molecular mass of TTHB246-BphJ chimeric complex, determined by static light scattering
152000
native molecular mass of TTHB246-TTHB247 complex, determined by static light scattering
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
enzyme can form a stable heterotetrameric complex with TTHB247 in vitro, consisting of two aldolase and two dehydrogenase subunits
heterotetramer
BphI-BphJ-complex, alpha2beta2, 2 * 32000 BphJ, 2 * 37000 BphI
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
half-life in complex with TTHB247 is 10 h; half-life of chimeric TTHB246-BphJ complex is 2.66 h for TTHB246 activity and 1.62 for BphJ activity; half-life of single enzyme is 42 h
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
purified enzyme can be stored at -80C, without loss of activity for at least 12 months
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
BphI and BphJ form a stable complex as they bind and coelute from Ni2+-NTA column, after purification N-terminal histidine tag of BphJ is proteolytically cleaved by thrombin digestion
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BphI and BphJ form a stable complex as they bind and coelute from Ni2+-NTA column, although only BphJ has the histidine tag. After purification, the N-terminal histidine tag of BphJ is proteolytically cleaved by thrombin digestion
purified to homogeneity using Ni2+-NTA chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
bphI and bphJ cloned into the plasmids pBTL4-T7 and pET28a
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bphI previous to mutagenesis cloned into plasmid pBTL4-T7, Escherichia coli
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chimeric complexes of Burkholderia xenovorans and Thermus thermophilus enzymes, TTHB246-BphJ and BphI-TTHB247 created by coexpression of the relevant genes in Escherichia coli using separate expression plasmids; separate expression of TTHB246 in recombinant Escherichia coli
coexpression of bphI and bphJ in Escherichia coli using two compatible plasmids (pBTL4 and pET28a) yielded soluble proteins
variants of BphI expressed in recombinant Escherichia coli BL21, lambdaDE3 cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G322A
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channels acetaldehyde with similar efficiency to wild-type, 30% lowered efficiency of isobutyraldehyde channeling
G322F
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unable to channel either acetaldehyde or propionaldehyde
G322L
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unable to channel either acetaldehyde or propionaldehyde
G323A
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channels acetaldehyde with similar efficiency to wild-type, unable to channel isobutyraldehyde
G323F
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unable to channel either acetaldehyde or propionaldehyde
G323L
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63% channeling efficiency for acetaldehyde, unable to channel propionaldehyde
H20S
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mutant generated by site-directed mutagenesis, pH profiles show the dependence of enzyme activity on hydroxide concentration
L87A
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mutant generated by site-directed mutagenesis, variant exhibits a 40fold preference for propionaldehyde over acetaldehyde in contrast to wild type with similar specificities for acetaldehyde and propionaldehyde
L87N
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mutant created by site-specific mutagenesis
L87N/Y290F
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exhibits stereoselectivity opposite to that of the wild type and alkyl chain length of the aldehyde does not affect stereochemical control
L87W
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mutant created by site-specific mutagenesis
L87W/Y290F
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exhibits stereoselectivity opposite to that of the wild type and alkyl chain length of the aldehyde does not affect stereochemical control
R16A
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mutant generated by site-directed mutagenesis, no detectable aldol cleavage and pyruvate R-proton exchange, supporting the role of Arg-16 in stabilizing a pyruvate enolate intermediate
Y290S
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mutant generated by site-directed mutagenesis, mutation results in a loss of stereochemical control as the variant is able to utilize substrates with R and S configurations at C4 with similar kinetic parameters
A324G
increase of channeling efficiency of propionaldehyde to a value comparable to that of acetaldehyde
additional information