Information on EC 2.3.1.B2 - type I polyhydroxybutyrate synthase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.3.1.B2
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
type I polyhydroxybutyrate synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
3-hydroxybutyryl-CoA + [(R)-3-hydroxybutanoate]n = [(R)-3-hydroxybutanoate]n+1 + CoA
show the reaction diagram
Tyr36 is catalytically essential
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SYSTEMATIC NAME
IUBMB Comments
acyl-CoA:3-hydroxybutyrate O-acyltransferase (short-chain)
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
class I enzyme
UniProt
Manually annotated by BRENDA team
class I enzyme
UniProt
Manually annotated by BRENDA team
uncultured bacterium from limestone soil
UniProt
Manually annotated by BRENDA team
uncultured bacterium from limestone soil
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
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PhaC is involved in the biosynthetic pathway for generation of (R)-3-hydroxybutyrate monomers from two acetyl-CoA molecules, and further of short-chain length polyhydroxyalkanoates, PHAs. The malonyl-CoA availability is a limiting factor to synthesis of poly(3-hydroxybutyrate), P(3HB), thus acetoacetyl-CoA synthetase, which is controlling the malonyl-CoA pool, is an important enzyme for increasing the P(3HB) production
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(R)-3-hydroxybutanoyl-CoA + [(R)-3-hydroxybutanoate]n
[(R)-3-hydroxybutanoate](n+1) + CoA
show the reaction diagram
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?
(R)-3-hydroxybutyryl-CoA + [(R)-3-hydroxybutanoate]n
[(R)-3-hydroxybutanoate](n+1) + CoA
show the reaction diagram
(R)-3-hydroxypentanoyl-CoA + [(R)-3-hydroxypentanoate]n
[(R)-3-hydroxypentoate](n+1)
show the reaction diagram
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activity is 10% of the reaction with 3-hydroxybutyryl-CoA
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-
?
3-hydroxyacyl-CoA + [(R)-3-hydroxyacyl]n
[(R)-3-hydroxyacyl]n+1 + CoA
show the reaction diagram
3-hydroxybutyryl-CoA + [(R)-3-hydroxyacyl]n
[(R)-3-hydroxyacyl]n+1 + CoA
show the reaction diagram
3-hydroxybutyryl-CoA + [(R)-3-hydroxybutanoate]n
[(R)-3-hydroxybutanoate](n+1) + CoA
show the reaction diagram
3-hydroxybutyryl-CoA + [(R)-3-hydroxybutanoate]n
[(R)-3-hydroxybutanoate]n+1 + CoA
show the reaction diagram
3-hydroxypropanoyl-CoA + (3-hydroxypropanoate)n
[(R)-3-hydroxypropanoate](n+1) + CoA
show the reaction diagram
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the value of the number-average molecular weight of the polymer obtained at a molar ratio of monomer-to-enzyme of 5000 is 25000 while the polydispersity is 4.7
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?
3-hydroxypropanoyl-CoA + 3-hydroxybutanoyl-CoA
poly(3-hydroxypropanoate-co-3-hydroxybutanoate) + CoA
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
3-hydroxyacyl-CoA + [(R)-3-hydroxyacyl]n
[(R)-3-hydroxyacyl]n+1 + CoA
show the reaction diagram
3-hydroxybutyryl-CoA + [(R)-3-hydroxybutanoate]n
[(R)-3-hydroxybutanoate]n+1 + CoA
show the reaction diagram
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
CoA
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very effective competitive inhibitor for the polymerization of 3-hydroxypropionyl-CoA
HBCH2CoA
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substrate analogue, in which the S-atom in hydroxybutanoyl-CoA is replaced with a CH2 group. HBCH2CoA is a competitive inhibitor of class I and class III PHB synthases. Upon incubation with a synthase acylated with a [3H]-saturated trimer-CoA, i.e. sTCoA, products are the methylene analogue of [3H]-sTCoA, [3H]-sT-CH2-CoA, saturated dimer-[3H]-sD-CO2H, and trimer-acid [3H]-sT-CO2H. HBCH2CoA may be reporting on the termination and repriming process of the synthases, rather than elongation
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.3
(R)-3-hydroxybutanoyl-CoA
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pH 7.2, 25C, presence of 0.05% Hecameg
0.19 - 0.32
(R)-3-hydroxybutyryl-CoA
0.189
3-hydroxypropionyl-CoA
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
196
(R)-3-hydroxybutanoyl-CoA
Cupriavidus necator
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pH 7.2, 25C, presence of 0.05% Hecameg
26 - 44
(R)-3-hydroxybutyryl-CoA
10
3-hydroxypropionyl-CoA
Cupriavidus necator
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.085
CoA
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0.04
HBCH2CoA
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pH 7.2, 25C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 8
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pooled monomeric and dimeric fractions of Strep2-PhaCRe, 30C, pH not specified in the publication
36
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purified recombinant Strep2-PhaCRe, 30C, pH not specified in the publication
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 66000, (His)6-tagged wild-type and G4D mutant PhaCRe, SDS-PAGE
monomer
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
one-step purification protocol
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recombinant
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recombinant N-terminally Strep2-tagged PhaC from Ralstonia eutropha by affinity chromatography. Strep2-PhaCRe co-purifies with the phasin protein, PhaP1, and with soluble polyhydroxybutyrate of 350 kDa MW in a high-molecular weight complex and in monomeric/dimeric forms with no associated PhaP1 or polyhydroxybutyrate
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
co-expression of PhaC with acetoacetyl-CoA synthetase, AACS, from Streptomyces sp. CL190 in Escherichia coli and Corynebacterium glutamicum leading to enhanced production of polyhydroxybutanoates, by cloning the AACS gene into the phaABC operon of Ralstonia eutropha
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construction of (His)6-tagged Ralstonia eutropha PHA synthase gene, expression in Escherichia coli
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construction of a recombinant Ralstonia eutropha PHB-4 harboring Aeromonas caviae biosynthesis genes under the control of a promoter for Ralstonia eutropha phb operon (phbRe promoter), and examination of the polyhydroxyalkanoate producing ability of the recombinants from various alkanoic acids as carbon sources. The polymerization step is not the rate-determining one in PHA biosynthesis by Ralstonia eutropha. The molecular weights of poly(3-hydroxybutyrate) produced by the recombinant strains are also independent of the levels of PHA synthase activity
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construction of an N-terminally Strep2-tagged PhaC, Strep2-PhaCRe, and integration into the Ralstonia eutropha genome in place of wild-type phaC and functional expression without a lag phase of CoA release in the enzyme reaction, functional expression of Strep2-PhaCRe in Escherichia coli strain BL21(DE3) showing a lag phase in CoA release
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expressed in Escherichia coli
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expression in Escherichia coli
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expression of wild-type and mutant enzymes in Escherichia coli altering its content of polyhydroxyalkanoates
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exypression in Escherichia coli
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mutant enzymes expressed in Escherichia coli
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the wild-type and mutated PHA synthase genes from Aeromonas caviae are introduced into Arabidopsis thaliana together with the NADPH-dependentacetoacetyl-CoA reductase gene from Ralstonia eutropha. Expression of the highly active mutated PHA synthase genes, N149S and D171G, leads to an 8-10fold increase in PHA content in the T1 transgenic Arabidopsis, compared to plants harboring the wild-type PHA synthase gene. In homozygous T2 progenies, PHA content is further increased up to 6.1 mg/g cell dry weight. GC/MS analysis of the purified PHA from the transformants revealed that these PHAs are poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymers consisting of 0.2-0.8 mol% 3-hydroxyvalerate
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wild-type and (His)6-tagged PhaCRe, expression in Escherichia coli. Wild-type enzyme expressed in Escherichia coli shows 35% of the enzyme from Ralstonia eutropha
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A372_C382del
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no detectable activity
A510S
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mutant is able to synthesize a lactate-3-hydroxybutanoate copolymer containing 7 mol% lactate and with a averge molecular weight of 320000 Da. The polymer contains a high ratio of an LA-LA-LA triad sequence
A510X
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mutation corresponds to position 481 in the class II lactate polymerizing polyhydroxyalkanoate synthase PhaC1PsSTQK, in which Gln481Lys is essential to its lactate polymerizing activity. Among 19 A510X mutants, 15 synthesize lactate-3-hydroxybutanoate copolymers
A81E
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in vitro activity is 108% of wild-type activity
A81G
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in vitro activity is 99% of wild-type activity
A81M
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in vitro activity is 101% of wild-type activity
A81P
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in vitro activity is 105% of wild-type activity
C438G
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no detectable activity
D281_D290del
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no detectable activity
D480N
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0.004% of the wild-type activity
E267K
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40% of wild-type activity
E578_A589del
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no detectable activity
F396L
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about 40% of the poly(3-hydroxybutyrate) content compared to wild-type
F420A
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poly(3-hydroxybutyrate) content is about 20% of wild-type value
F420C
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poly(3-hydroxybutyrate) content is about 35% of wild-type value
f420D
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poly(3-hydroxybutyrate) content is about 20% of wild-type value
F420E
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poly(3-hydroxybutyrate) content is about 10% of wild-type value
F420G
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poly(3-hydroxybutyrate) content is about 20% of wild-type value
F420H
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poly(3-hydroxybutyrate) content is about 35% of wild-type value
F420I
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poly(3-hydroxybutyrate) content is about 30% of wild-type value
F420K
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poly(3-hydroxybutyrate) content is about 35% of wild-type value
F420L
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poly(3-hydroxybutyrate) content is about 35% of wild-type value
F420M
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poly(3-hydroxybutyrate) content is about 40% of wild-type value
F420N
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poly(3-hydroxybutyrate) content is about 45% of wild-type value
F420Q
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poly(3-hydroxybutyrate) content is about 30% of wild-type value
F420R
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poly(3-hydroxybutyrate) content is about 30% of wild-type value
F420S
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F420S enzyme has a significant decrease in its lag phase compared to that of the wild-type enzyme. Poly(3-hydroxybutyrate) content is about 85% of wild-type value
F420T
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poly(3-hydroxybutyrate) content is about 35% of wild-type value
F420V
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poly(3-hydroxybutyrate) content is about 35% of wild-type value
F420W
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poly(3-hydroxybutyrate) content is about 25% of wild-type value
F420Y
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poly(3-hydroxybutyrate) content is about 35% of wild-type value
G4A
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 14% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4C
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 24% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4D
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 58% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4D/F420S
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mutant shows a higher poly(3-hydroxybutyrate) content and in vivo concentration of PhaCRe enzyme than the F420S mutant, the molecular weight of the poly(3-hydroxybutyrate) polymer of the double mutant is similar to that of the F420S mutant
G4E
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 58% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4F
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 45% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4H
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 56% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4I
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 56% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has rather similar molecular weights with that of the wild-type
G4K
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 58% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4L
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 2% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4M
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 24% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4N
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 57% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4P
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 54% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4Q
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 55% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has rather similar molecular weights with that of the wild-type
G4R
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 54% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4S
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 56% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has rather similar molecular weights with that of the wild-type
G4T
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 56% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has rather similar molecular weights with that of the wild-type
G4V
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 12% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4W
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 13% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
G4Y
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poly(3-hydroxybutyrate) content of Escherichia coli harboring mutant PhaCRe is 54% of Ralstonia eutropha wild-type value. Poly(3-hydroxybutyrate) produced by mutant has higher molecular weights with that of the wild-type
H481Q
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20% of the wild-type activity
H508Q
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less than 0.0005% of the wild-type activity
L446K
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15% of wild-type activity, no change in substrate specificity
N208D
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about 40% of the poly(3-hydroxybutyrate) content compared to wild-type
T231I
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no detectable activity
T323S
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no detectable activity
V585_A589del
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no detectable activity
Y445F
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38% of wild-type activity, no change in substrate specificity
Y75E
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in vitro activity is 137% of wild-type activity
Y75E/A81E
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in vitro activity is 154% of wild-type activity
Y75F
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in vitro activity is 104% of wild-type activity
Y75F/A81M
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in vitro activity is 105% of wild-type activity
Y75G
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in vitro activity is 110% of wild-type activity
Y75G/A81G
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in vitro activity is 119% of wild-type activity
Y75P
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in vitro activity is 138% of wild-type activity
Y75P/A81P
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in vitro activity is 162% of wild-type activity
A510S
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mutant is able to synthesize a lactate-3-hydroxybutanoate copolymer containing 7 mol% lactate and with a averge molecular weight of 320000 Da. The polymer contains a high ratio of an LA-LA-LA triad sequence
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A510X
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mutation corresponds to position 481 in the class II lactate polymerizing polyhydroxyalkanoate synthase PhaC1PsSTQK, in which Gln481Lys is essential to its lactate polymerizing activity. Among 19 A510X mutants, 15 synthesize lactate-3-hydroxybutanoate copolymers
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C319A
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site-directed mutagenesis
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E130D/S325T/S477G/Q481K
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engineered polyhydroxybutanoate synthase able to accept 2-hydroxyacyl-CoAs as substrates
Q481K
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
Q481K/Q508L
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
Q481M
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
Q481M/Q508L
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
Q481R
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
Q481R/Q508L
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
Q508L
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
S325T
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
S325T/Q508L
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
S477R
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
S477R/Q508L
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
Q481K
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
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Q481M
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
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Q481R
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
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Q508L
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
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S325T
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site-directed mutagenesis, the mutant shows an altered substrate specificity shifted to short-chain acyl-CoAs, corresponding to the reaction of a type I PHA synthase, compared to the wild-type enzyme, which is more specific for medium-chain substrates as a type II PHA synthase
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E130D/S325T/S477G/Q481K
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engineered polyhydroxybutanoate synthase able to accept 2-hydroxyacyl-CoAs as substrates
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis