Information on EC 5.4.99.64 - 2-hydroxyisobutanoyl-CoA mutase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
5.4.99.64
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RECOMMENDED NAME
GeneOntology No.
2-hydroxyisobutanoyl-CoA mutase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2-hydroxy-2-methylpropanoyl-CoA = (S)-3-hydroxybutanoyl-CoA
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
methyl tert-butyl ether degradation
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2-methylpropene degradation
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SYSTEMATIC NAME
IUBMB Comments
2-hydroxy-2-methylpropanoyl-CoA mutase
The enzyme, characterized from the bacterium Aquincola tertiaricarbonis, uses radical chemistry to rearrange the positions of both a methyl group and a hydroxyl group. It consists of two subunits, the smaller one containing a cobalamin cofactor. It plays a central role in the degradation of assorted substrates containing a tert-butyl moiety.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
D5WTR7 i.e. subunit RcmA, D5WTR8 i.e. subunit RcmB
D5WTR7 and D5WTR8
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
D5WTR7 and D5WTR8
mutase from strain DSM 2912 catalyzes isomerization of (R)-3-hydroxybutanoyl-CoA about 7000 times more efficiently than the mutase from Aquincola tertiaricarbonis L108. The most striking structural difference between the two mutases, likely determining stereospecificity, is a replacement of active-site residue Asp found in strain L108 at position 117 with Val in the enzyme from strain DSM 2912, resulting in a reversed polarity at this binding site
metabolism
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
2-hydroxy-2-methylpropanoyl-CoA
show the reaction diagram
(R)-3-hydroxybutanoyl-CoA
2-hydroxybutanoyl-CoA
show the reaction diagram
D5WTR7 and D5WTR8
catalytic efficiency for the (R)-enantiomer is about 11 times higher than for the (S)-enantiomer
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r
(S)-3-hydroxybutanoyl-CoA
2-hydroxy-2-methylpropanoyl-CoA
show the reaction diagram
(S)-3-hydroxybutanoyl-CoA
2-hydroxybutanoyl-CoA
show the reaction diagram
D5WTR7 and D5WTR8
catalytic efficiency for the (R)-enantiomer is about 11 times higher than for the (S)-enantiomer
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r
2,2-dimethylpropanoyl-CoA
?
show the reaction diagram
I3VE77 and I3VE74
wild-type enzyme as well as I90V (HcmA) and I90A (HcmA) mutant enzymes can also isomerize pivaloyl-CoA and isovaleryl-CoA, albeit at more than 10times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA
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r
2-hydroxy-2-methylpropanoyl-CoA
(S)-3-hydroxybutanoyl-CoA
show the reaction diagram
2-hydroxyisobutanoyl-CoA
(S)-3-hydroxyisobutanoyl-CoA + (R)-3-hydroxyisobutanoyl-CoA
show the reaction diagram
D5WTR7 and D5WTR8
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r
butanoyl-CoA
isobutanoyl-CoA
show the reaction diagram
I3VE77 and I3VE74
low rearrangement activity, corresponding to approximately 2% of the (S)-3-hydroxybutanoyl-CoA conversion rate
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r
isobutanoyl-CoA
butanoyl-CoA
show the reaction diagram
isovaleryl-CoA
?
show the reaction diagram
I3VE77 and I3VE74
wild-type enzyme as well as I90V (HcmA) and I90A (HcmA) mutant enzymes can also isomerize pivalyl- and isovaleryl-CoA, albeit at more than 10times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA
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?
isovaleryl-CoA
pentanoyl-CoA
show the reaction diagram
I3VE77 and I3VE74
at 10 times lower rate than substrate (S)-3-hydroxybutanoyl-CoA
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r
methylmalonyl-CoA
succinyl-CoA
show the reaction diagram
I3VE77 and I3VE74
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r
pivalyl-CoA
?
show the reaction diagram
I3VE77 and I3VE74
at 10 times lower rate than substrate (S)-3-hydroxybutanoyl-CoA
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r
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
2-hydroxy-2-methylpropanoyl-CoA
(S)-3-hydroxybutanoyl-CoA
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cobalamin
I3VE77 and I3VE74
the smaller one of the two subunits (hcmB) contains a cobalamin cofactor
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0075 - 1.66
(R)-3-hydroxybutanoyl-CoA
0.0146 - 1.76
(S)-3-hydroxybutanoyl-CoA
0.0435 - 0.977
2,2-dimethylpropanoyl-CoA
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0.042 - 1.84
2-hydroxy-2-methylpropanoyl-CoA
0.09
2-hydroxyisobutanoyl-CoA
D5WTR7 and D5WTR8
pH 7.8, 55C
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3.34
Butanoyl-CoA
I3VE77 and I3VE74
pH 6.6, 30C, wild-type enzyme
0.55
isobutanoyl-CoA
I3VE77 and I3VE74
pH 6.6, 30C, wild-type enzyme
0.147 - 0.329
isovaleryl-CoA
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.003 - 4.9
(R)-3-hydroxybutanoyl-CoA
0.002 - 1.6
(S)-3-hydroxybutanoyl-CoA
0.013 - 0.14
2,2-dimethylpropanoyl-CoA
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0.0067 - 0.05
2-hydroxy-2-methylpropanoyl-CoA
3.7
2-hydroxyisobutanoyl-CoA
D5WTR7 and D5WTR8
pH 7.8, 55C
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0.0033
Butanoyl-CoA
I3VE77 and I3VE74
pH 6.6, 30C, wild-type enzyme
0.001
isobutanoyl-CoA
I3VE77 and I3VE74
pH 6.6, 30C, wild-type enzyme
0.009 - 0.011
isovaleryl-CoA
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00199 - 56.7
(R)-3-hydroxybutanoyl-CoA
0.0047 - 5
(S)-3-hydroxybutanoyl-CoA
0.017 - 3.2
2,2-dimethylpropanoyl-CoA
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0.011 - 1.053
2-hydroxy-2-methylpropanoyl-CoA
40
2-hydroxyisobutanoyl-CoA
D5WTR7 and D5WTR8
pH 7.8, 55C
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0.0099
Butanoyl-CoA
I3VE77 and I3VE74
pH 6.6, 30C, wild-type enzyme
0.0018
isobutanoyl-CoA
I3VE77 and I3VE74
pH 6.6, 30C, wild-type enzyme
0.028 - 0.062
isovaleryl-CoA
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.8
D5WTR7 and D5WTR8
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.2 - 7
I3VE77 and I3VE74
pH 6.2: about 60% of maximal activity, pH 7.0: about 60% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
D5WTR7 and D5WTR8
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 35
I3VE77 and I3VE74
25C: about 50% pf maxim al activity, 35C: about 45% of maximal activity
30
D5WTR7 and D5WTR8
25% of maximum activity
75
D5WTR7 and D5WTR8
55% of maximum activity
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
14515
I3VE77 and I3VE74
1 * 63414 + 1 * 14515, calculated from sequence, the enzyme consists of a large acyl-CoA-binding and a small B12-binding subunit, HcmA and HcmB, respectively
63414
I3VE77 and I3VE74
1 * 63414 + 1 * 14515, calculated from sequence, the enzyme consists of a large acyl-CoA-binding and a small B12-binding subunit, HcmA and HcmB, respectively
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
multimer
D5WTR7 and D5WTR8
x * subunit RcmA + x * subunit RcmB
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure in complex with coenzyme B12 and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. subunit HcmA residues IleA90 and AspA117. AspA117 determine the orientation of the hydroxyl group of the acyl-CoA esters by H-bond formation, thus determining stereospecificity of catalysis; crystal structure of the enzyme in complex with coenzyme B12 and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. Crystals are grown in 16% PEG 3350, 0.1 M Tris,pH 7.5, by the hanging-drop vapor diffusion method at 4C
I3VE77 and I3VE74
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
-
I3VE77 and I3VE74
expression in Escherichia coli
D5WTR7 and D5WTR8
wild-type mutase genes are cloned in Escherichia coli strains
I3VE77 and I3VE74
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D117A
I3VE77 and I3VE74
mutation in subunit HcmA, results in significantly increased activity toward (R)-3-hydroxybutyryl-CoA; mutation in the large subunit HcmA results in significantly increased activity toward (R)-3-hydroxybutyryl-CoA
D117V
I3VE77 and I3VE74
mutation in subunit HcmA, results in significantly increased activity toward (R)-3-hydroxybutyryl-CoA and high activity toward pivalyl-CoA; mutation in the large subunit HcmA results in significantly increased activity toward (R)-3-hydroxybutyryl-CoA. The mutant enzyme shows high activity toward pivalyl-CoA
I90A
I3VE77 and I3VE74
mutation in the large subunit HcmA results in a significant reduction in conversion rates and diminution of the catalytic efficiencies for the main substrates of the wild-type enzyme, (S)-3-hydroxybutyryl-coA and 2-hydroxyisobutyryl-CoA. Wild-type enzyme as well as mutant enzyme can also isomerize pivalyl- and isovaleryl-CoA, albeit at more than 10times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA; strong decrease in activity
I90F
I3VE77 and I3VE74
complete loss of enzyme activity
I90L
I3VE77 and I3VE74
mutation in the large subunit HcmA results in a significant reduction in conversion rates and diminution of the catalytic efficiencies for the main substrates of the wild-type enzyme, (S)-3-hydroxybutyryl-coA and 2-hydroxyisobutyryl-CoA. Mutant enzyme does not show any significant activities with pivalyl-CoA and isovaleryl-CoA; strong decrease in activity
I90Y
I3VE77 and I3VE74
complete loss of enzyme activity
I90F
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complete loss of enzyme activity
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I90V
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significant activities are only obtained with (S)-3-hydroxybutanoyl- and 2-hydroxyisobutanoyl-CoA, showing less than 20% of the wild-type rates. The substitution I90V results in Km values close to 2 mM and a nearly 100fold diminution of the catalytic efficiency with (S)-3-hydroxybuytryl-CoA
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I90Y
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complete loss of enzyme activity
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis