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Information on EC 5.4.99.13 - isobutyryl-CoA mutase
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5.4.99.13 isobutyryl-CoA mutaseIUBMB Comments
This bacterial enzyme utilizes 5'-deoxyadenosylcobalamin as a cofactor. Following substrate binding, the enzyme catalyses the homolytic cleavage of the cobalt-carbon bond of AdoCbl, yielding cob(II)alamin and a 5'-deoxyadenosyl radical, which initiates the the carbon skeleton rearrangement reaction by hydrogen atom abstraction from the substrate. At the end of each catalytic cycle the 5'-deoxyadenosyl radical and cob(II)alamin recombine, regenerating the resting form of the cofactor. The enzyme is prone to inactivation resulting from occassional loss of the 5'-deoxyadenosyl molecule. Inactivated enzymes are repaired by the action of EC 2.5.1.17, cob(I)yrinic acid a,c-diamide adenosyltransferase, and a G-protein chaperone, which restore cob(II)alamin (which is first reduced to cob(I)alamin by an unidentified reductase) to 5'-deoxyadenosylcobalamin and load it back on the mutase. Some mutases are fused with their G-protein chaperone. These enzyme can also catalyse the interconversion of isovaleryl-CoA with pivalyl-CoA.
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Word Map
- 5.4.99.13
- methylmalonyl-coa
-
b12-dependent
- mutases
- acyl-coas
- n-butyryl-coa
- cinnamonensis
-
icmfs
- polyketide
- succinyl-coa
- monensin
- isomerases
- meaa
-
5'-deoxyadenosylcobalamin-dependent
- 2-hydroxyisobutyrate
- extorquens
- isobutyrate
-
cobiialamin
-
2r-methylmalonyl-coa
- synthesis
-
b12-binding
-
mutase-catalyzed
- 5'-deoxyadenosine
-
5\'-deoxyadenosyl
-
coa-dependent
- isobutanol
-
methylmalonyl
- methylobacterium
- crotonyl-coa
-
pivalic
- biofuel production
The enzyme appears in viruses and cellular organisms
Synonyms
isobutyryl-coa mutase, butyryl-coa:isobutyryl-coa mutase, pivalyl-coa mutase, adocbl-dependent pcm, isovaleryl-coa/pivalyl-coa mutase, isobutyryl-coa mutase fused, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
adenosylcobalamin-dependent isobutyryl-CoA mutase
AdoCbl-dependent PCM
butyryl-CoA:isobutyryl-CoA mutase
-
-
-
-
ICM
IcmF
isobutyryl coenzyme A mutase
-
-
-
-
isobutyryl-CoA mutase fused
PCM
pivalyl-CoA mutase
IcmF
-
a fusion between isobutyryl-CoA mutase and its G-protein chaperone
IcmF
-
a fusion between isobutyryl-CoA mutase and its G-protein chaperone
isobutyryl-CoA mutase fused
-
a fusion between isobutyryl-CoA mutase and its G-protein chaperone
isobutyryl-CoA mutase fused
-
a fusion between isobutyryl-CoA mutase and its G-protein chaperone
isobutyryl-CoA mutase fused
-
a fusion between isobutyryl-CoA mutase and its G-protein chaperone
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2-methylpropanoyl-CoA = butanoyl-CoA
loss of stereocontrol during the reaction
-
2-methylpropanoyl-CoA = butanoyl-CoA
either a random or ordered sequential mechanism
2-methylpropanoyl-CoA = butanoyl-CoA
loss of stereocontrol during the reaction
Streptomyces virginiae A3823.5
-
-
2-methylpropanoyl-CoA = butanoyl-CoA
either a random or ordered sequential mechanism
Streptomyces virginiae A3823.5
-
-
2-methylpropanoyl-CoA = butanoyl-CoA
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
intramolecular transfer reaction
-
-
-
-
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SYSTEMATIC NAME
IUBMB Comments
2-methylpropanoyl-CoA CoA-carbonylmutase
This bacterial enzyme utilizes 5'-deoxyadenosylcobalamin as a cofactor. Following substrate binding, the enzyme catalyses the homolytic cleavage of the cobalt-carbon bond of AdoCbl, yielding cob(II)alamin and a 5'-deoxyadenosyl radical, which initiates the the carbon skeleton rearrangement reaction by hydrogen atom abstraction from the substrate. At the end of each catalytic cycle the 5'-deoxyadenosyl radical and cob(II)alamin recombine, regenerating the resting form of the cofactor. The enzyme is prone to inactivation resulting from occassional loss of the 5'-deoxyadenosyl molecule. Inactivated enzymes are repaired by the action of EC 2.5.1.17, cob(I)yrinic acid a,c-diamide adenosyltransferase, and a G-protein chaperone, which restore cob(II)alamin (which is first reduced to cob(I)alamin by an unidentified reductase) to 5'-deoxyadenosylcobalamin and load it back on the mutase. Some mutases are fused with their G-protein chaperone. These enzyme can also catalyse the interconversion of isovaleryl-CoA with pivalyl-CoA.
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CAS REGISTRY NUMBER
COMMENTARY
116405-23-3
-
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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
2-methylpropanoyl-CoA
butanoyl-CoA
interconversion
-
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
interconversion, preferred substrate
-
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
interconversion
-
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
interconversion
-
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
interconversion, preferred substrate
-
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
-
valine and fatty acid catabolism
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
valine and fatty acid catabolism
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
-
biosynthesis of the antibiotic monesin-A
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r
2-methylpropanoyl-CoA
butanoyl-CoA
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biosynthesis of the antibiotic monesin-A
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r
2-methylpropanoyl-CoA
butanoyl-CoA
-
biosynthesis of the antibiotic monesin-A
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
biosynthesis of the antibiotic monesin-A
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
Streptomyces virginiae A3823.5
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biosynthesis of the antibiotic monesin-A
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r
2-methylpropanoyl-CoA
butanoyl-CoA
Streptomyces virginiae A3823.5
valine and fatty acid catabolism
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
Streptomyces virginiae A3823.5
biosynthesis of the antibiotic monesin-A
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r
isobutyrylcarba(dethia)-CoA
butyrylcarba(dethia)-CoA
-
stable toward hydrolysis facilitating estimation of the equilibrium constant
stable toward hydrolysis facilitating estimation of the equilibrium constant
r
additional information
?
-
enzyme substrate specificity, active site architecture and determinants of substrate specificity, comparison of the substrate-bound structures to that of substrate-free holo-IcmF-GDP, overview. Acyl-CoA substrates are threaded through the TIM barrel substrate-binding domain
-
-
?
additional information
?
-
enzyme substrate specificity, active site architecture and determinants of substrate specificity, comparison of the substrate-bound structures to that of substrate-free holo-IcmF-GDP, overview. Acyl-CoA substrates are threaded through the TIM barrel substrate-binding domain
-
-
?
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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
2-methylpropanoyl-CoA
butanoyl-CoA
interconversion
-
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
interconversion
-
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
interconversion
-
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
-
valine and fatty acid catabolism
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
valine and fatty acid catabolism
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
-
biosynthesis of the antibiotic monesin-A
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
-
biosynthesis of the antibiotic monesin-A
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
-
biosynthesis of the antibiotic monesin-A
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
biosynthesis of the antibiotic monesin-A
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
Streptomyces virginiae A3823.5
-
biosynthesis of the antibiotic monesin-A
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
Streptomyces virginiae A3823.5
valine and fatty acid catabolism
-
r
2-methylpropanoyl-CoA
butanoyl-CoA
Streptomyces virginiae A3823.5
biosynthesis of the antibiotic monesin-A
-
r
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
the GTPase activity of IcmF powers the ejection of the inactive cob(II)alamin cofactor and requires the presence of an acceptor protein, adenosyltransferase, for receiving it. Adenosyltransferase in turn converts cob(II)alamin to AdoCbl in the presence of ATP and a reductant. The repaired cofactor is then reloaded onto IcmF in a GTPase-gated step. Transfer of inactive cob(II)alamin from ATR to IcmF is disfavored. The mechanistic details of cofactor loading and offloading from the AdoCbl-dependent IcmF are distinct from those of the homologous methylmalonyl-CoA mutase/G-protein chaperone system. The nonhydrolyzable analogue, GMPPCP, weakens the affinity of IcmF for AdoCbl by about 60fold and allows loading of only one of two cofactor-binding sites. AdoCbl binding in the presence of GMPPCP is primarily enthalpically driven. The slight inhibition of AdoCbl transfer in the presence of GTP or GDP is likely due to the higher affinity of ATR for AdoCbl in the presence of these nucleotides, while ATP enhances the transfer. kinetics, overview
-
5'-deoxyadenosylcobalamin
AdoCbl, required, affinity for AdoCbl is unaffected by the presence of GDP
5'-deoxyadenosylcobalamin
AdoCbl, required, the active sites in the CmIcmF dimer exhibit identical affinity for AdoCbl. Addition of isovaleryl-CoA to wild-type holo-IcmF results in formation of the intermediate cob(II)alamin. AdoCbl is quantitatively converted to cob(II)alamin when isovaleryl-CoA is added to F598A or to the F598I/L or Q742A mutants inactivating the enzyme. Cob(II)alamin formation in activates the enzyme
5'-deoxyadenosylcobalamin
AdoCbl, required, two conformations: C3'-endo and C2'-endo, conformational change of the 5'-deoxyadenosyl group from C2'-endo to C3'-endo contributes to initiation of catalysis. Adenosylcobalamin (coenzyme B12) is an organometallic enzyme cofactor for radical chemistry. Its reactivity is based on a unique covalent cobalt-carbon (Co-C) bond that is sufficiently weak to allow for reversible homolytic cleavage in enzyme active sites, generating a 5'-deoxyadenosyl radical in the presence of an appropriate substrate. The radical can then abstract a substrate hydrogen atom and initiate difficult chemical transformations
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Co2+
-
or cobalamin, strictly required. During growth on limiting concentrations of cobalt, strain accumulates 2-hydroxyisobutyrate
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
GDP
-
the presence of GDP decreases kcat about 1.6-1.7fold while increasing the Km about 2fold. Consequently, the catalytic efficiency kcat/Km value of IcmF decreases 3fold in the presence of GDP
GTP
-
the presence of GTP decreases kcat about 1.6-1.7fold while increasing the Km about 2fold. Consequently, the catalytic efficiency kcat/Km value of IcmF decreases 4.5fold in the presence of GTP
additional information
inactivation rates of OH2Cbl formation for wild-type and mutant enzymes, overview
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
the pivalyl-CoA mutase activity of engineered PCM shows a sigmoidal dependence on the concentration of isolvaleryl-CoA
-
0.36
2-methylpropanoyl-CoA
recombinant mutant F598I, pH 7.5, 37°C
0.45
2-methylpropanoyl-CoA
recombinant mutant F598A, pH 7.5, 37°C
0.67
2-methylpropanoyl-CoA
recombinant mutant F598V, pH 7.5, 37°C
0.79
2-methylpropanoyl-CoA
recombinant mutant Q742A, pH 7.5, 37°C
0.89
2-methylpropanoyl-CoA
recombinant mutant F598L, pH 7.5, 37°C
1.1
2-methylpropanoyl-CoA
recombinant wild-type enzyme, pH 7.5, 37°C
2.4
2-methylpropanoyl-CoA
recombinant mutant Q742N, pH 7.5, 37°C
0.0453
isobutyryl-CoA
-
recombinant enzyme, in the presence of GDP, in 50 mM sodium phosphate buffer, pH 7.5, 250 mM NaCl at 37°C
0.0508
isobutyryl-CoA
-
recombinant enzyme, in the presence of GTP, in 50 mM sodium phosphate buffer, pH 7.5, 250 mM NaCl at 37°C
0.2
isovaleryl-CoA
recombinant engineered PCM-F mutant, pH 7.5, 37°C
0.37
isovaleryl-CoA
recombinant engineered PCM mutant, pH 7.5, 37°C
1.4
isovaleryl-CoA
recombinant wild-type enzyme, pH 7.5, 37°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.08
2-methylpropanoyl-CoA
recombinant mutant F598A, pH 7.5, 37°C
0.08
2-methylpropanoyl-CoA
recombinant mutant Q742A, pH 7.5, 37°C
0.14
2-methylpropanoyl-CoA
recombinant mutant F598I, pH 7.5, 37°C
0.2
2-methylpropanoyl-CoA
recombinant mutant F598V, pH 7.5, 37°C
1.27
2-methylpropanoyl-CoA
recombinant mutant F598L, pH 7.5, 37°C
1.83
2-methylpropanoyl-CoA
recombinant mutant Q742N, pH 7.5, 37°C
48.33
2-methylpropanoyl-CoA
recombinant wild-type enzyme, pH 7.5, 37°C
1.88
isobutyryl-CoA
-
recombinant enzyme, in the presence of GTP, in 50 mM sodium phosphate buffer, pH 7.5, 250 mM NaCl at 37°C
1.96
isobutyryl-CoA
-
recombinant enzyme, in the presence of GDP, in 50 mM sodium phosphate buffer, pH 7.5, 250 mM NaCl at 37°C
3.1
isobutyryl-CoA
-
recombinant enzyme, in 50 mM sodium phosphate buffer, pH 7.5, 250 mM NaCl at 37°C
0.0036
isovaleryl-CoA
recombinant mutant F598I, pH 7.5, 37°C
0.0084
isovaleryl-CoA
recombinant mutant F598L, pH 7.5, 37°C
0.014
isovaleryl-CoA
recombinant engineered PCM mutant, pH 7.5, 37°C
0.023
isovaleryl-CoA
recombinant engineered PCM-F mutant, pH 7.5, 37°C
0.062
isovaleryl-CoA
recombinant wild-type enzyme, pH 7.5, 37°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.101
2-methylpropanoyl-CoA
recombinant mutant Q742A, pH 7.5, 37°C
0.18
2-methylpropanoyl-CoA
recombinant mutant F598A, pH 7.5, 37°C
0.299
2-methylpropanoyl-CoA
recombinant mutant F598V, pH 7.5, 37°C
0.39
2-methylpropanoyl-CoA
recombinant mutant F598I, pH 7.5, 37°C
0.76
2-methylpropanoyl-CoA
recombinant mutant Q742N, pH 7.5, 37°C
1.43
2-methylpropanoyl-CoA
recombinant mutant F598L, pH 7.5, 37°C
43.94
2-methylpropanoyl-CoA
recombinant wild-type enzyme, pH 7.5, 37°C
37
isobutyryl-CoA
-
recombinant enzyme, in the presence of GTP, in 50 mM sodium phosphate buffer, pH 7.5, 250 mM NaCl at 37°C
43
isobutyryl-CoA
-
recombinant enzyme, in the presence of GDP, in 50 mM sodium phosphate buffer, pH 7.5, 250 mM NaCl at 37°C
0.0062
isovaleryl-CoA
recombinant mutant F598I, pH 7.5, 37°C
0.0242
isovaleryl-CoA
recombinant mutant F598V, pH 7.5, 37°C
0.0378
isovaleryl-CoA
recombinant engineered PCM mutant, pH 7.5, 37°C
0.0431
isovaleryl-CoA
recombinant mutant F598A, pH 7.5, 37°C
0.0443
isovaleryl-CoA
recombinant wild-type enzyme, pH 7.5, 37°C
0.115
isovaleryl-CoA
recombinant engineered PCM-F mutant, pH 7.5, 37°C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.023
-
with butanoyl-CoA as the substrate from extracts of Streptomyces cinnamonensis
0.18
purified recombinant enzyme, pivalyl-CoA mutase activity of engineered PCM, pH 7.5, 37°C
1
-
activity reconstituted by mixing recombinant His6-IcmA produced in Escherichia coli with IcmB isolated from Streptomyces cinnamonensis
3.3
the specific activity varies with the molar ratio of IcmA to IcmB
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.4
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Streptomyces virginiae A3823.5
isolated from methyl t-butyl ether-contaminated groundwater, able to grow on methyl t-butyl ether and ethyl t-butyl ether as sole carbon sources
-
-
brenda
isolated from methyl t-butyl ether-contaminated groundwater, able to grow on methyl t-butyl ether and ethyl t-butyl ether as sole carbon sources
-
-
brenda
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
physiological function
additional information
evolution
enzyme IcmF belongs to the family of adenosylcobalamin-dependent isomerases, whose members catalyze carbon skeleton rearrangements using radical chemistry
evolution
-
enzyme IcmF belongs to the family of adenosylcobalamin-dependent isomerases, whose members catalyze carbon skeleton rearrangements using radical chemistry
-
physiological function
IcmF is a 5'-deoxyadenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the carbon skeleton rearrangement of isobutyryl-CoA to butyryl-CoA. It is a bifunctional protein resulting from the fusion of a G-protein chaperone with GTPase activity and the cofactor- and substrate-binding mutase domains with isomerase activity. IcmF is prone to inactivation during catalytic turnover, thus setting up its dependence on a cofactor repair system. The GTPase activity of IcmF powers the ejection of the inactive cob(II)alamin cofactor and requires the presence of an acceptor protein, adenosyltransferase, for receiving it. Adenosyltransferase in turn converts cob(II)alamin to AdoCbl in the presence of ATP and a reductant. The repaired cofactor is then reloaded onto IcmF in a GTPase-gated step. The mechanistic details of cofactor loading and offloading from the AdoCbl-dependent IcmF are distinct from those of the homologous methylmalonyl-CoA mutase/G-protein chaperone system, overview
physiological function
the isobutyryl-CoA mutase variant, IcmF, catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA and it also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation
physiological function
-
the isobutyryl-CoA mutase variant, IcmF, catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA and it also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation
-
physiological function
-
IcmF is a 5'-deoxyadenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the carbon skeleton rearrangement of isobutyryl-CoA to butyryl-CoA. It is a bifunctional protein resulting from the fusion of a G-protein chaperone with GTPase activity and the cofactor- and substrate-binding mutase domains with isomerase activity. IcmF is prone to inactivation during catalytic turnover, thus setting up its dependence on a cofactor repair system. The GTPase activity of IcmF powers the ejection of the inactive cob(II)alamin cofactor and requires the presence of an acceptor protein, adenosyltransferase, for receiving it. Adenosyltransferase in turn converts cob(II)alamin to AdoCbl in the presence of ATP and a reductant. The repaired cofactor is then reloaded onto IcmF in a GTPase-gated step. The mechanistic details of cofactor loading and offloading from the AdoCbl-dependent IcmF are distinct from those of the homologous methylmalonyl-CoA mutase/G-protein chaperone system, overview
-
additional information
enzyme IcmF in general is a fusion between the radical B12 enzyme isobutyryl-CoA mutase and its G-protein chaperone. IcmF from Cupriavidus metallidurans, which also contains a G-protein domain in addition to the mutase domains, with AdoCbl in the ICM active site and GDP-Mg2+ in the G-protein active site (holo-IcmF-GDP)
additional information
GDP binding by the enzyme is accompanied by favorable enthalpic and entropic changes and is unaffected by 5'-deoxyadenosylcobalamin
additional information
IcmF is a natural variant in which the small and large subunits found in ICM are fused via a middle G-protein chaperone domain, active site sequence comparisons, overview
additional information
-
IcmF is a natural variant in which the small and large subunits found in ICM are fused via a middle G-protein chaperone domain, active site sequence comparisons, overview
-
additional information
-
enzyme IcmF in general is a fusion between the radical B12 enzyme isobutyryl-CoA mutase and its G-protein chaperone. IcmF from Cupriavidus metallidurans, which also contains a G-protein domain in addition to the mutase domains, with AdoCbl in the ICM active site and GDP-Mg2+ in the G-protein active site (holo-IcmF-GDP)
-
additional information
-
GDP binding by the enzyme is accompanied by favorable enthalpic and entropic changes and is unaffected by 5'-deoxyadenosylcobalamin
-
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
135000
recombinant detagged enzyme, large subunit dimer, gel filtration
14300
alpha2beta2, IcmA2IcmB2, 2 * 62500 + 2 * 14300, gel filtration, IcmB provides the cobalamin-binding domain
20000
recombinant detagged enzyme, small subunit monomer, gel filtration
62500
alpha2beta2, IcmA2IcmB2, 2 * 62500 + 2 * 14300, gel filtration, IcmB provides the cobalamin-binding domain
additional information
size exclusion chromatography yields an estimated molecular mass of 135 kDa, consistent with the large subunit being a homodimer. The recombinant small subunit of PCM is purified as N-terminally His6-tagged protein and elutes with an apparent mass of 20 kDa in gel filtration, suggesting that it exists as a monomer based on the predicted mass of the polypeptide of 17.7 kDa. Gel filtration of a 1:1 mixture of the large and small subunits ofthe enzyme in presence of 5'-deoxyadenosylcobalamin, shows no evidence of complex formation, indicating weak interaction between the subunits under these conditions
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
homodimer
monomer
tetramer
additional information
homodimer
2 * 62300, recombinant detagged enzyme large subunit, SDS-PAGE
homodimer
-
2 * 62300, recombinant detagged enzyme large subunit, SDS-PAGE
-
monomer
1 * 17700, about, recombinant detagged enzyme small subunit, sequence calculation
monomer
-
1 * 17700, about, recombinant detagged enzyme small subunit, sequence calculation
-
tetramer
alpha2beta2, IcmA2IcmB2, 2 * 62500 + 2 * 14300, gel filtration, IcmB provides the cobalamin-binding domain
tetramer
Streptomyces virginiae A3823.5
-
alpha2beta2, IcmA2IcmB2, 2 * 62500 + 2 * 14300, gel filtration, IcmB provides the cobalamin-binding domain
-
additional information
IcmF is a natural variant in which the small and large subunits found in ICM are fused via a middle G-protein chaperone domain, domain structure and sequence comparisons, overview
additional information
-
IcmF is a natural variant in which the small and large subunits found in ICM are fused via a middle G-protein chaperone domain, domain structure and sequence comparisons, overview
-
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified enzyme in complex with four different substrates, GDP and isovaleryl-CoA, or isobutyryl-CoA or n-butytryl-CoA or pivalyl-CoA, hanging drop vapor diffusion technique, mixing of 0.001 ml of 11.7 mg/ml IcmF protein in 100 mM NaCl, 50 mM HEPES, pH 7.5, 1 mM GDP, 3 mM MgCl2, 0.3 mM AdoCbl, with 0.001 ml of reservoir solution containing 0.7-0.75 M potassium sodium tartrate, 0.2 M ammonium acetate, 0.1 M imidazole, pH 7.0-7.7, and 3% v/v ethylene glycol, and equilibration against 0.5 ml reservoir solution, 3-6 weeks, at 25°C, X-ray diffraction structure determination and analysis at 3.4-3.5 A resolution
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
F598A
site-directed mutagenesis, the mutation causes a 17fold increase in catalytic efficiency in pivalyl-CoA mutase activity and a concomitant about 240fold decrease in isobutyryl-CoA mutase activity compared to wild-type IcmF. The mutation of the single residue in IcmF tunes the substrate specificity to an about 4000fold increase in the specificity for the unnatural substrate. The F598A mutant is more susceptible to inactivation than wild-type IcmF
F598I
site-directed mutagenesis, the isobutyryl-CoA mutase activity of the mutant is diminished and the isovaleryl-CoA mutase activity is increased compared to wild-type
F598L
site-directed mutagenesis, the isobutyryl-CoA mutase activity of the mutant is diminished and the isovaleryl-CoA mutase activity is increased compared to wild-type
F598V
site-directed mutagenesis, the isobutyryl-CoA mutase activity of the mutant is diminished and the isovaleryl-CoA mutase activity is increased compared to wild-type
Q742A
site-directed mutagenesis, the mutant shows undetectable isovaleryl-CoA mutase activity and significantly diminished isobutyryl-CoA mutase activity compared to wild-type
Q742N
site-directed mutagenesis, the mutant shows undetectable isovaleryl-CoA mutase activity and significantly diminished isobutyryl-CoA mutase activity compared to wild-type
F598A
-
site-directed mutagenesis, the mutation causes a 17fold increase in catalytic efficiency in pivalyl-CoA mutase activity and a concomitant about 240fold decrease in isobutyryl-CoA mutase activity compared to wild-type IcmF. The mutation of the single residue in IcmF tunes the substrate specificity to an about 4000fold increase in the specificity for the unnatural substrate. The F598A mutant is more susceptible to inactivation than wild-type IcmF
-
F598I
-
site-directed mutagenesis, the isobutyryl-CoA mutase activity of the mutant is diminished and the isovaleryl-CoA mutase activity is increased compared to wild-type
-
additional information
additional information
AdoCbl is quantitatively converted to cob(II)alamin when isovaleryl-CoA is added to F598A or to the F598I/L or Q742A mutants inactivating the enzyme, The F598A mutant inactivates 3fold more rapidly than wild-type IcmF
additional information
engineering of enzyme IcmF to increase pivalyl-CoA mutase activityby targeting two active site residues predicted to be key to substrate selectivity based on sequence alignments and crystal structures. PCM-F is an artificially engineered variant of PCM (IcmF) in which the large and small subunits are fused via an 11-amino acid linker
additional information
-
AdoCbl is quantitatively converted to cob(II)alamin when isovaleryl-CoA is added to F598A or to the F598I/L or Q742A mutants inactivating the enzyme, The F598A mutant inactivates 3fold more rapidly than wild-type IcmF
-
additional information
-
engineering of enzyme IcmF to increase pivalyl-CoA mutase activityby targeting two active site residues predicted to be key to substrate selectivity based on sequence alignments and crystal structures. PCM-F is an artificially engineered variant of PCM (IcmF) in which the large and small subunits are fused via an 11-amino acid linker
-
additional information
-
the production of isobutanol, a branched-chain alcohol that can be used as a gasoline substitute, using a CoA-dependent pathway in recombinant Ralstonia eutropha strain H16. The designed pathway is constituted of three modules: chain elongation, rearrangement, and modification. First, the chain elongation and modification modules are integrated and optimized, esulting in the production of about 200 mg/l n-butanol from fructose or 30 mg/L from formate by engineered Ralstonia eutropha. Subsequently, the rearrangement module is incorporated, featuring native isobutyryl-CoA mutase in Ralstonia eutropha. The engineered strain produces about 30 mg/l isobutanol from fructose. The demonstrated carbon skeleton rearrangement chemistry may be used to expand the range of the chemicals accessible with CoA-dependent pathways
additional information
-
disruption of IcmA in Streptomyces cinnamonensis
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni-NTA-Sepharose column chromatography, Source 15Q column chromatography, and Superdex 200 gel filtration
partial, using ammonium sulfate precipitation, and DEAE-Sepharose column chromatography
-
recombinant His-MBP-tagged wild-type enzyme and His6-SUMO-tagged mutant enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, tag cleavage by TEV protease, dialysis, amylose affinity chromatography (only wild-type), and gel filtration
recombinant IcmB is purified using Q-Sepharose, and two Mono Q column chromatography
using ammonium sulfate precipitation, DEAE, Q-sepharose, superdex, gel electrophoresis, and B12 affinity column chromatography
-
Ni-NTA-Sepharose column chromatography, Source 15Q column chromatography, and Superdex 200 gel filtration
-
Ni-NTA-Sepharose column chromatography, Source 15Q column chromatography, and Superdex 200 gel filtration
-
Ni-NTA-Sepharose column chromatography, Source 15Q column chromatography, and Superdex 200 gel filtration
-
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
gene icmF, recombinant expression of N-terminally His-tagged IcmF
gene icmF, recombinant expression of soluble His-MBP-tagged wild-type enzyme and His6-SUMO-tagged mutant enzyme in Escherichia coli strain BL21(DE3)
IcmA, expression in Escherichia coli and Streptomyces lividans, IcmA shows 70% of homology to the methylmalonyl-CoA mutase large subunit from Streptomyces cinnamonensis
-
IcmB, expression in Escherichia coli, IcmB shows significant identity to several cobalamin-dependent proteins
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
biofuel production
-
the production of isobutanol, a branched-chain alcohol that can be used as a gasoline substitute, using a CoA-dependent pathway in recombinant Ralstonia eutropha strain H16. The designed pathway involves isobutyryl-CoA mutase activity. The engineered strain produces about 30 mg/l isobutanol from fructose
synthesis
synthesis
IcmF is potentially useful as a reagent for bioremediation of pivalic acid found in pharmaceutical wastewaters (pivalic acid esters are used as pro-drugs) and/or for the biosynthesis of a simple beta-nonfunctional alpha-quaternary carboxylic acid. Another potential application of IcmF is in metabolic engineering of pathways to produce branched C4 and C5 building blocks that can subsequently be converted to useful derivatives, for example, the corresponding isobutyl and pivalyl alcohols
synthesis
-
the production of isobutanol, a branched-chain alcohol that can be used as a gasoline substitute, using a CoA-dependent pathway in recombinant Ralstonia eutropha strain H16. The designed pathway involves isobutyryl-CoA mutase. The engineered strain produces about 30 mg/l isobutanol from fructose. The carbon skeleton rearrangement chemistry demonstrated may be used to expand the range of the chemicals accessible with CoA-dependent pathways
synthesis
-
IcmF is potentially useful as a reagent for bioremediation of pivalic acid found in pharmaceutical wastewaters (pivalic acid esters are used as pro-drugs) and/or for the biosynthesis of a simple beta-nonfunctional alpha-quaternary carboxylic acid. Another potential application of IcmF is in metabolic engineering of pathways to produce branched C4 and C5 building blocks that can subsequently be converted to useful derivatives, for example, the corresponding isobutyl and pivalyl alcohols
-
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Brendelberger, G.; Retey, J.; Ashworth, D.M.; Reynolds, K.; Willenbrock, F.; Robinson, J.A.
Die enzymatische Umwandlung von Isobutyryl- zu n-Butyrylcarba(dethia)-Coenzym A: eine CoenzymB12-abhngige Geruestumlagerung
Angew. Chem.
100
1122-1125
1988
Streptomyces virginiae
-
brenda
Matthies, C.; Schink, B.
Reciprocal isomerization of butyrate and isobutyrate by the strictly anaerobic bacterium strain WoG13 and methanogenic isobutyrate degradation by a defined triculture
Appl. Environ. Microbiol.
58
1435-1439
1992
Streptomyces virginiae
brenda
Moore, B.S.; Eisenberg, R.; Weber, C.; Bridges, A.; Nanz, D.; Robinson, J.A.
On the stereospecificity of the coenzyme B12-dependent isobutyryl-CoA mutase reaction
J. Am. Chem. Soc.
117
11285-11291
1995
Streptomyces virginiae, Streptomyces virginiae A3823.5
-
brenda
Ratnatilleke, A.; Vrijbloed, J.W.; Robinson, J.A.
Cloning and sequencing of the coenzyme B(12)-binding domain of isobutyryl-CoA mutase from Streptomyces cinnamonensis, reconstitution of mutase activity, and characterization of the recombinant enzyme produced in Escherichia coli
J. Biol. Chem.
274
31679-31685
1999
Streptomyces virginiae (Q9RJ84), Streptomyces virginiae A3823.5 (Q9RJ84)
brenda
Zerbe-Burkhardt, K.; Ratnatilleke, A.; Philippon, N.; Birch, A.; Leiser, A.; Vrijbloed, J.W.; Hess, D.; Hunziker, P.; Robinson, J.A.
Cloning, sequencing, expression, and insertional inactivation of the gene for the large subunit of the coenzyme B12-dependent isobutyryl-CoA mutase from Streptomyces cinnamonensis
J. Biol. Chem.
273
6508-6517
1998
Streptomyces virginiae
brenda
Rohwerder, T.; Breuer, U.; Benndorf, D.; Lechner, U.; Mueller, R.H.
The alkyl tert-butyl ether intermediate 2-hydroxyisobutyrate is degraded via a novel cobalamin-dependent mutase pathway
Appl. Environ. Microbiol.
72
4128-4135
2006
uncultured bacterium, uncultured bacterium L108
brenda
Cracan, V.; Padovani, D.; Banerjee, R.
IcmF is a fusion between the radical B12 enzyme isobutyryl-CoA mutase and its G-protein chaperone
J. Biol. Chem.
285
655-666
2010
Paraburkholderia xenovorans, Geobacillus kaustophilus, Nocardia farcinica
brenda
Black, W.B.; Zhang, L.; Kamoku, C.; Liao, J.C.; Li, H.
Rearrangement of coenzyme A-acylated carbon chain enables synthesis of isobutanol via a novel pathway in Ralstonia eutropha
ACS Synth. Biol.
7
794-800
2018
Cupriavidus necator
brenda
Kitanishi, K.; Cracan, V.; Banerjee, R.
Engineered and native coenzyme B12-dependent isovaleryl-CoA/pivalyl-CoA mutase
J. Biol. Chem.
290
20466-20476
2015
Cupriavidus metallidurans (Q1LRY0), Cupriavidus metallidurans DSM 2839 (Q1LRY0)
brenda
Jost, M.; Born, D.A.; Cracan, V.; Banerjee, R.; Drennan, C.L.
Structural basis for substrate specificity in adenosylcobalamin-dependent isobutyryl-CoA mutase and related acyl-CoA mutases
J. Biol. Chem.
290
26882-26898
2015
Cupriavidus metallidurans (Q1LRY0), Cupriavidus metallidurans ATCC 43123 (Q1LRY0)
brenda
Li, Z.; Kitanishi, K.; Twahir, U.T.; Cracan, V.; Chapman, D.; Warncke, K.; Banerjee, R.
Cofactor editing by the G-protein metallochaperone domain regulates the radical B
J. Biol. Chem.
292
3977-3987
2017
Cupriavidus metallidurans (Q1LRY0), Cupriavidus metallidurans ATCC 43123 (Q1LRY0)
brenda
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