Information on EC 2.3.1.B4 - type III polyhydroxybutyrate synthase

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The expected taxonomic range for this enzyme is: Bacteria, Archaea

EC NUMBER
COMMENTARY hide
2.3.1.B4
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
type III polyhydroxybutyrate synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
3-hydroxyacyl-CoA + [(R)-3-hydroxyacyl]n = [(R)-3-hydroxyacyl]n+1 + CoA
show the reaction diagram
can use short- and medium-chain acyl-CoA with a chain length of 3-14 carbon atoms. The enzyme is composed of two nonidentical subunits.
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
butanoate fermentation
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SYSTEMATIC NAME
IUBMB Comments
acyl-CoA:3-hydroxybutyrate O-acyltransferase (short- and medium-chain)
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SAG 1403-2
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Manually annotated by BRENDA team
SAG 1403-2
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
PhaC; PCC 6912
SwissProt
Manually annotated by BRENDA team
Cyanothece sp.
Gloeocapsa sp.
strain PCC 7428
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-
Manually annotated by BRENDA team
28 strains belonging to 15 genera in the family Halobacteriaceae
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Manually annotated by BRENDA team
subunit PhaE and PhaC
I3R9Z3 and I3R9Z4
SwissProt
Manually annotated by BRENDA team
gene is a biomarker for microorganisms that contain class III PHA synthase
UniProt
Manually annotated by BRENDA team
gene is a biomarker for microorganisms that contain class III PHA synthase
UniProt
Manually annotated by BRENDA team
no activity in Cyanothece sp.
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Manually annotated by BRENDA team
no activity in Cyanothece sp. PCC 8955
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Manually annotated by BRENDA team
no activity in Gloeothece membranacea
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Manually annotated by BRENDA team
no activity in Gloeothece membranacea PCC 6501
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Manually annotated by BRENDA team
no activity in Stanieria cyanosphaera
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Manually annotated by BRENDA team
no activity in Stanieria cyanosphaera PCC 7437
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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28 strains belonging to 15 genera in the family Halobacteriaceae, sequence comparisons, phylogenetic analysis, and analyses of conserved regions of type III PHA synthases, overview
physiological function
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PHA accumulation rates in the differnt strains harboring type III PHA synthases, overview
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-hydroxybutyryl-CoA + [3-hydroxybutanoate]n
[(R)-3-hydroxybutanoate](n+1) + CoA
show the reaction diagram
Q9F5P8 and Q9F5P9, Q9F5P9
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-
-
?
(R)-3-hydroxypentanoyl-CoA + [(R)-3-hydroxypentanoate]n
[(R)-3-hydroxypentanoate](n+1)
show the reaction diagram
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
3-hydroxybutyryl-CoA + [(R)-3-hydroxybutanoate]n
[(R)-3-hydroxybutyrate](n+1) + CoA
show the reaction diagram
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?
3-hydroxybutyryl-CoA + [3-hydroxybutanoate]n
[3-hydroxybutanoate](n+1) + CoA
show the reaction diagram
Q9F5P8 and Q9F5P9, Q9F5P9
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-
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?
3-hydroxyvaleryl-CoA + [3-hydroxyvalerate]n
[3-hydroxyvalerate](n+1) + CoA
show the reaction diagram
Q9F5P8 and Q9F5P9, Q9F5P9
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-
-
?
4-hydroxybutyryl-CoA + [3-hydroxybutanoate]n
[4-hydroxybutanoate](n+1) + CoA
show the reaction diagram
Q9F5P8 and Q9F5P9, Q9F5P9
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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
additional information
?
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I3R9Z3 and I3R9Z4
the enzyme is responsible for synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
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
0.063 - 0.45
(R)-3-hydroxybutyryl-CoA
0.18 - 0.32
(R)-3-hydroxypentanoyl-CoA
0.03
(R)-3-hydroxyvaleryl-CoA
Q9F5P8 and Q9F5P9, Q9F5P9
;
0.085
4-hydroxybutyryl-CoA
Q9F5P8 and Q9F5P9, Q9F5P9
;
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
18.67 - 320
(R)-3-hydroxybutyryl-CoA
1.43 - 8
(R)-3-hydroxypentanoyl-CoA
122
(R)-3-hydroxyvaleryl-CoA
Ectothiorhodospira shaposhnikovii
Q9F5P8 and Q9F5P9, Q9F5P9
based on the assumption that 2 PhaC and 2 PhaE subunits comprise one catalytic site; based on the assumption that 2 PhaC and 2 PhaE subunits comprise one catalytic site
297
4-hydroxybutyryl-CoA
Ectothiorhodospira shaposhnikovii
Q9F5P8 and Q9F5P9, Q9F5P9
based on the assumption that 2 PhaC and 2 PhaE subunits comprise one catalytic site; based on the assumption that 2 PhaC and 2 PhaE subunits comprise one catalytic site
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.014
HBCH2CoA
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pH 7.2, 37C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.8
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in sodium phosphate as well as in Tris/HCl buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8.8
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pH 7.0: about 40% of maximal activity, pH 8.8: about 65% of maximal activity, Tris/HCl buffer
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
I3R9Z3 and I3R9Z4
PhaEHme and PhaCHme proteins are strongly bound to the granules
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
390000
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non-denaturing PAGE
400000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 39320, calculated
dodecamer
Q9F5P8 and Q9F5P9, Q9F5P9
6 * 41700 (PhaE) + 6 * 40600 (PhaC), two major complexes are identified in preparations of purified PHA synthase. The large complex appears to be composed of 12 PhaC subunits and 12 PhaE subunits (dodecamer). The small complex appears to be composed of 6 PhaC and 6 PhaE subunits (hexamer). In dilute aqueous solution, the synthase is predominantly composed of hexamer and has low activity accompanied with a significant lag period at the initial stage of reaction. The percentage of dodecameric complex increases with increasing salt concentration. The dodecameric complex has a greatly increased specific activity for the polymerization of (R)-3-hydroxybutyryl-CoA and a negligible lag period. The PHA synthase from Ectothiorhodospira shaposhnikoVii may catalyze a living polymerization and two PhaC and two PhaE subunits may comprise a single catalytic site in the synthase complex, SDS-PAGE; 6 * 41700 (PhaE) + 6 * 40600 (PhaC), two major complexes are identified in preparations of purified PHA synthase. The large complex appears to be composed of 12 PhaC subunits and 12 PhaE subunits (dodecamer). The small complex appears to be composed of 6 PhaC and 6 PhaE subunits (hexamer). In dilute aqueous solution, the synthase is predominantly composed of hexamer and has low activity accompanied with a significant lag period at the initial stage of reaction. The percentage of dodecameric complex increases with increasing salt concentration. The dodecameric complex has a greatly increased specific activity for the polymerization of (R)-3-hydroxybutyryl-CoA and a negligible lag period. The PHA synthase from Ectothiorhodospira shaposhnikoVii may catalyze a living polymerization and two PhaC and two PhaE subunits may comprise a single catalytic site in the synthase complex, SDS-PAGE
hexamer
Q9F5P8 and Q9F5P9, Q9F5P9
3 * 41700 (PhaE) + 3 * 40600 (PhaC), two major complexes are identified in preparations of purified PHA synthase. The large complex appears to be composed of 12 PhaC subunits and 12 PhaE subunits (dodecamer). The small complex appears to be composed of 6 PhaC and 6 PhaE subunits (hexamer). In dilute aqueous solution, the synthase is predominantly composed of hexamer and has low activity accompanied with a significant lag period at the initial stage of reaction. The percentage of dodecameric complex increases with increasing salt concentration. The dodecameric complex has a greatly increased specific activity for the polymerization of (R)-3-hydroxybutyryl-CoA and a negligible lag period. The PHA synthase from Ectothiorhodospira shaposhnikoVii may catalyze a living polymerization and two PhaC and two PhaE subunits may comprise a single catalytic site in the synthase complex, SDS-PAGE; 3 * 41700 (PhaE) + 3 * 40600 (PhaC), two major complexes are identified in preparations of purified PHA synthase. The large complex appears to be composed of 12 PhaC subunits and 12 PhaE subunits (dodecamer). The small complex appears to be composed of 6 PhaC and 6 PhaE subunits (hexamer). In dilute aqueous solution, the synthase is predominantly composed of hexamer and has low activity accompanied with a significant lag period at the initial stage of reaction. The percentage of dodecameric complex increases with increasing salt concentration. The dodecameric complex has a greatly increased specific activity for the polymerization of (R)-3-hydroxybutyryl-CoA and a negligible lag period. The PHA synthase from Ectothiorhodospira shaposhnikoVii may catalyze a living polymerization and two PhaC and two PhaE subunits may comprise a single catalytic site in the synthase complex, SDS-PAGE
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
sequence has potential N-glycosylation, myristoylation, protein kinase C and casein kinase II type phosphorylation domains
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 37
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purified PHA synthase exhibits no significant loss of specific activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
loss of specific activity of the PHA synthase is observed during purification at 4C
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
each subunit was cloned, expressed, and purified as a (His)6-tagged construct
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recombinant His-tagged PhaECAv from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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recombinantly expressed in Escherichia coli; recombinantly expressed in Escherichia coli
Q9F5P8 and Q9F5P9, Q9F5P9
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
28 strains belonging to 15 genera in the family Halobacteriaceae, sequence comparisons, phylogenetic analysis, overview
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a central region of the phaC gene of Cyanothece sp. strain PCC 8303 is cloned, sequenced and heterologously expressed in Escherichia coli; a central region of the phaC gene of Cyanothece sp. strain PCC 8303 is cloned, sequenced and heterologously expressed in Escherichia coli
Cyanothece sp.
cloning of the gene cluster (phaECHme) encoding a polyhydroxyalkanoate (PHA) synthase in Haloferax mediterranei CGMCC 1.2087 via thermal asymmetric interlaced PCR
I3R9Z3 and I3R9Z4
constructs of phaCDm alone (pBBRMCS-2::phaCDm) and of phaEDmCDm (pBBRMCS-2::phaEDmCDm) in various vectors are obtained and transferred to several strains of Escherichia coli, as well as to the PHA-negative mutants PHB-4 and GPp104 of Ralstonia eutropha and Pseudomonas putida, respectively. In cells of the recombinant strains harboring phaEDmCDm small but significant amounts (up to 1.7% of cell dry matter) of poly(3-hydroxybutyrate) and of PHA synthase activity (up to 1.5 U/mg protein) are detected. Hybrid synthases consisting of PhaCDm and PhaE of Thiococcus pfennigii or Synechocystis sp. strain PCC 6308 are also constructed and are shown to be functionally active
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each subunit was cloned, expressed, and purified as a (His)6-tagged construct
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entire PHA synthase structural gene of Chlorogloeopsis fritschii PCC 6912 is cloned, sequenced and heterologously expressed in Escherichia coli
entire PHA synthase structural gene of Synechococcus sp. strain MA19 is cloned, sequenced and heterologously expressed in Escherichia coli; entire PHA synthase structural gene of Synechococcus sp. strain MA19 is cloned, sequenced and heterologously expressed in Escherichia coli
in vivo studies on a Wautersia eutropha strain in which the class I synthase gene has been replaced with the D302A-PhaCPhaE gene and the organism examined under PHB production conditions by transmission electron microscopy. Very small granules are observed in contrast to the 200-500 nm granules observed with the wild-type strain
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PHA synthase genes (phaC and phaE) are cloned by screening a genomic library for PHA accumulation in Escherichia coli cells; PHA synthase genes (phaC and phaE) are cloned by screening a genomic library for PHA accumulation in Escherichia coli cells
Q9F5P8 and Q9F5P9, Q9F5P9
recombinant expression of His-tagged PhaECAv in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C130S
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121% of wild-type activity in the initial phase of reaction, 16% of wild-type activity in tghe second phase of reaction
C149A
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inactive mutant protein of PhaC
C149S
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0.1% of wild-type activity in the initial phase of reaction, 0.09% of wild-type activity in tghe second phase of reaction
c149S/H331Q
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no activity
C292A
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mutant of PhaC with wild-type activity
D302A
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incubation of D302A-PhaCPhaE with [14C]-hydroxybutanoyl-CoA results in detection of oligomeric HBs covalently bound to PhaC, at hydroxybutanoyl-CoA to enzyme ratios between 5 and 100
D302N
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0.012% of wild-type activity in the initial phase of reaction, 0.29% of wild-type activity in tghe second phase of reaction
H303Q
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0.25% of wild-type activity in the initial phase of reaction, 1.6% of wild-type activity in tghe second phase of reaction
H331Q
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51% of wild-type activity in the initial phase of reaction, 73% of wild-type activity in tghe second phase of reaction
additional information
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the enzyme is used for in vitro synthesis of polyhydroxyalkanoates on a hydrophobic support, i.e. highly oriented pyrolytic graphite. Using PhaECAv and 3-hydroxyoctanoyl-CoA at room temperature, a poly(3-hydroxyoctanoate) [P(3HO)] film is formed on the hydrophobic support with a thickness of a few nanometers, as revealed by atomic force microscopy
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis