Information on EC 2.3.3.14 - homocitrate synthase

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

EC NUMBER
COMMENTARY hide
2.3.3.14
-
RECOMMENDED NAME
GeneOntology No.
homocitrate synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
acetyl-CoA + H2O + 2-oxoglutarate = (2R)-2-hydroxybutane-1,2,4-tricarboxylate + CoA
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
aldol condensation
condensation
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
coenzyme B biosynthesis
-
-
FeMo cofactor biosynthesis
-
-
L-lysine biosynthesis IV
-
-
L-lysine biosynthesis V
-
-
lysine metabolism
-
-
Lysine biosynthesis
-
-
Pyruvate metabolism
-
-
Metabolic pathways
-
-
Microbial metabolism in diverse environments
-
-
Biosynthesis of antibiotics
-
-
SYSTEMATIC NAME
IUBMB Comments
acetyl-CoA:2-oxoglutarate C-acetyltransferase (thioester-hydrolysing, carboxymethyl forming)
Belongs in the alpha-aminoadipate pathway of lysine synthesis, along with EC 4.2.1.36, homoaconitate hydratase. The enzyme also acts with oxaloacetate as substrate, but more slowly [2,3].
CAS REGISTRY NUMBER
COMMENTARY hide
9075-60-9
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
formerly Aspergillus fumigatus
UniProt
Manually annotated by BRENDA team
formerly Aspergillus fumigatus
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
wild type strain STA-4 and lysine-requiring auxotrophic strains 33933 and mutant STL-7
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
Saccharomycopsis lipolytica
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
-
the enzyme is linked to the key process of DNA damage repair through the essential MYST family histone acetyltransferase Esa1 and the H2A.Z. The enzyme has a role in addition to amino acid synthesis, and functions in nuclear activities involving chromatin regulation that are distinct from its previously established role in lysine biosynthesis
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acetyl-CoA + H2O + 2-oxoadipate
2-hydroxyhexane-1,2,6-tricarboxylate
show the reaction diagram
-
-
-
-
?
acetyl-CoA + H2O + 2-oxoglutarate
(2R)-2-hydroxybutane-1,2,4-tricarboxylate + CoA
show the reaction diagram
acetyl-CoA + H2O + 2-oxoglutarate
(R)-2-hydroxybutane-1,2,4-tricarboxylate + CoA
show the reaction diagram
acetyl-CoA + H2O + 2-oxoglutarate
2-hydroxybutane-1,2,4-tricarboxylate + CoA
show the reaction diagram
acetyl-CoA + H2O + oxaloacetate
2-hydroxypropane-1,2,3-tricarboxylate + CoA
show the reaction diagram
acetyl-CoA + H2O + oxaloacetate
?
show the reaction diagram
-
oxaloacetate is a slow alternative substrate
-
-
?
acetyl-CoA + H2O + oxaloacetate
? + CoA
show the reaction diagram
-
oxaloacetate is a substrate with lower affinity than 2-oxoglutarate
-
-
?
additional information
?
-
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
acetyl-CoA + H2O + 2-oxoglutarate
(2R)-2-hydroxybutane-1,2,4-tricarboxylate + CoA
show the reaction diagram
acetyl-CoA + H2O + 2-oxoglutarate
(R)-2-hydroxybutane-1,2,4-tricarboxylate + CoA
show the reaction diagram
acetyl-CoA + H2O + 2-oxoglutarate
2-hydroxybutane-1,2,4-tricarboxylate + CoA
show the reaction diagram
additional information
?
-
-
homocitrate synthase is a labile target for oxidative stress caused by CuZn-superoxide dismutase depletion
-
-
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
acetyl-CoA
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mn2+
-
activates
Zinc
-
bound in the active site
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
-
0.01 mM, 91% inhibition
2,2'-dipyridyl
-
0.01 mM, 82% inhibition
2-aminoethyl-L-cysteine
-
-
-
2-methylcitrate
89.6% residual activity at 5 mM
2-oxoglutarate
-
-
2-Oxomalonate
-
-
acetyl-CoA
-
-
AMP
-
1 mM, 13-15% inhibition
benzylpenicillin
-
20 mM, partial inhibition is enhanced by lysine
Ca2+
96.3% residual activity at 1 mM
casein hydrolysate
-
about 50% inhibition
-
citrate
desulfo-CoA
-
uncompetitive
dipicolinic acid
Saccharomycopsis lipolytica
-
non-competitive inhibitor of 2-oxoglutarate fixation
dipyridyl
95% residual activity at 0.1 mM
DL-4,5-transdehydrolysine
Saccharomycopsis lipolytica
-
5 mM, 90% inhibition
DL-5-hydroxylysine
-
-
DL-allo-5-Hydroxylysine
Saccharomycopsis lipolytica
-
5 mM, 75% inhibition
DL-alpha-aminoadipate
-
-
DL-alpha-Difluoromethyllysine
-
strong inhibitor of isoform Lys22p, but not Lys21p
DL-Homolysine
-
inhibition of enzyme from wild-type strain, enzyme from S-(beta-aminoethyl)-L-cysteine resistant mutant strain is not inhibited
glyoxylate
-
competitive, analysis of pH-dependence of inhibition
hydroxylysine
iodoacetic acid
-
0.01 mM, 71% inhibition
K+
91.7% residual activity at 0.1 mM
L-Arg
L-lysine
L-norleucine
-
-
LL-Diaminopimelic acid
Saccharomycopsis lipolytica
-
5 mM, 90% inhibition
lysine
Mg2+
-
10 mM MgSO4, 30% inhibition
Na2HAsO4
-
0.01 mM, 67% inhibition
Na2SO4
90.9% residual activity at 0.2 mM
NaF
-
0.01 mM, 73% inhibition
NEM
-
0.1 mM, 90% inhibition
oxalglycine
-
competitive inhibition versus 2-oxoglutarate
oxaloacetate
-
-
oxalylglycine
-
-
p-hydroxymercuribenzoate
-
0.0001 mM, complete inhibition
pipecolic acid
Saccharomycopsis lipolytica
-
non-competitive inhibition on the fixation of both substrates
pyridine-2,3-dicarboxylate
-
-
Pyridine-2,4-dicarboxylate
-
-
Pyridine-2,5-dicarboxylate
-
-
pyridine-2,6-dicarboxylate
-
-
pyridine-2-carboxylate
-
-
S-(2-aminoethylo)-L-cysteine
-
-
-
S-(beta-aminoethyl)-L-cysteine
-
1.1 mM required for half-maximal inhibition of enzyme from wild-type strain, enzyme from S-(beta-aminoethyl)-L-cysteine resistant mutant strain is not inhibited
Selenalysine
-
7.5 mM, 90% inhibition, half-maximal inhibition at 1.9 mM, competitive inhibition against both acetyl-CoA and 2-oxoglutarate
succinyl phosphonate
-
-
thialysine
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
L-lysine
-
stimulates activity on histone H4
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.24
2-oxoadipate
-
-
0.0054 - 42
2-oxoglutarate
0.006 - 0.8
acetyl-CoA
0.255 - 2.28
oxaloacetate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.182 - 5.133
2-oxoglutarate
0.18 - 4.983
acetyl-CoA
0.967
oxaloacetate
Thermus thermophilus
-
-
additional information
additional information
Saccharomyces cerevisiae
-
the kcat for E155Q decreases at high pH, similar to the wild type enzyme, but is pH independent at low pH
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
26
2-oxoglutarate
-
pH 7.2
10
2-Oxomalonate
-
pH 7.2
13.5 - 200
citrate
0.008 - 0.11
CoA
0.014 - 0.18
desulfo-CoA
5.1
DL-alpha-aminoadipate
-
-
26
Glutarate
-
pH 7.2
0.4
glyoxylate
-
pH 7.2
0.008 - 0.0094
L-Lys
5
L-lysine
-
-
0.94 - 15
L-thialysine
0.053 - 2
lysine
1
oxalate
-
pH 7.2
0.9
oxalglycine
-
in 50 mM HEPES, pH 7.5, at 25C
4
oxaloacetate
-
pH 7.2
7.7
oxalylglycine
-
pH 7.2
3
pyridine-2,3-dicarboxylate
-
pH 7.2
0.6
Pyridine-2,4-dicarboxylate
-
pH 7.2
14
Pyridine-2,5-dicarboxylate
-
pH 7.2
0.7
pyridine-2,6-dicarboxylate
-
pH 7.2
0.43
pyridine-2-carboxylate
-
pH 7.2
0.14 - 1.1
Selenalysine
46
succinyl phosphonate
-
pH 7.2
additional information
additional information
-
-
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
16.5
2-aminoethyl-L-cysteine
Candida albicans
-
isoform Lys21p, at pH 7.5 and 22C
-
5.7
DL-5-hydroxylysine
Candida albicans
-
isoform Lys22p, at pH 7.5 and 22C
0.032
DL-alpha-Difluoromethyllysine
Candida albicans
-
isoform Lys22p, at pH 7.5 and 22C
18.7
L-Arg
Candida albicans
-
isoform Lys22p, at pH 7.5 and 22C
0.0541 - 0.286
L-Lys
1.8
S-(2-aminoethylo)-L-cysteine
Candida albicans
-
isoform Lys22p, at pH 7.5 and 22C
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0
-
no activity without additive
0.0003
-
additive CuCl2
0.0008
-
additive FeSO4
0.0013
-
additive ZnSO4
0.0036
-
additive NiCl2
0.0075
-
additive CaCl2
0.0333
-
additive CoCl2
0.0392
-
additive MnCl2
0.0415
-
additive MgSO4
0.8
purified enzyme, in 200 mM HEPES, pH 7.5, at 22C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 8
7.5 - 8.2
Saccharomycopsis lipolytica
-
-
8 - 8.7
-
-
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
-
activity assay at room temperature
37
-
wild-type strain
43
-
enzyme from S-(beta-aminoethyl)-L-cysteine resistant mutant strain
50
-
activity assay
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 40
-
25C: about 60% of maximal activity, 40C: about 50% of maximal activity
40 - 80
-
40C: about 70% of maximal activity, 80C: about 60% of maximal activity
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
mainly located in
Manually annotated by BRENDA team
-
25% of total activity
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
58600
theoretical
60000
-
gel filtration
66000
different bands are detected at 66 kDa (monomer), 148 kDa (dimer), 226 kDa (trimer), and 280 kDa (tetramer), and the dimeric and tetrameric compositions are most abundant, gel filtration
89000
-
gel filtration
148000
different bands are detected at 66 kDa (monomer), 148 kDa (dimer), 226 kDa (trimer), and 280 kDa (tetramer), and the dimeric and tetrameric compositions are most abundant, gel filtration
155000
-
gel filtration
200700
-
isoform Lys22p, gel filtration
210300
-
isoform Lys21p, gel filtration
226000
different bands are detected at 66 kDa (monomer), 148 kDa (dimer), 226 kDa (trimer), and 280 kDa (tetramer), and the dimeric and tetrameric compositions are most abundant, gel filtration
280000
different bands are detected at 66 kDa (monomer), 148 kDa (dimer), 226 kDa (trimer), and 280 kDa (tetramer), and the dimeric and tetrameric compositions are most abundant, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotetramer
-
4 * 47087, isoform Lys22p, calculated from amino acid sequence; 4 * 48300, isoform Lys21p, SDS-PAGE; 4 * 48314, isoform Lys21p, calculated from amino acid sequence; 4 * 49400, isoform Lys22p, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
the crystal structure of the homocitrate synthase apoenzyme and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate are reported
-
the structure of SpHCS in complex with the inhibitor L-lysine and Zn2+ is solved at a resolution of 2.38 A
the crystal structures of homocitrate synthase complexed with 2-oxoglutarate, (R)-2-hydroxybutane-1,2,4-tricarboxylate, or lysine are determined
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 11
the enzyme retains 70% residual activity after 6 h at pH 4.0, 100% activity after 6 h at pH 5.0-6.0, 80% residual activity after 6.5 h at pH 7.0-9.0, and 50% residual activity after 50 min at pH 11.0
719384
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0 - 60
the enzyme retains 100% activity after more than 8 h at 0-37C, 70% residual activity after 270 min at 42C, and 50% residual activity after 2 min at 60C
70
-
rapid loss of activity above
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme is very unstable in crude and protamine sulphate-treated desalted extracts
-
freezing and thawing results in 50% loss of activity
-
the purified enzyme can be stabilized using a combination of guanidine hydrochloride, alpha-cyclodextrin and ammonium sulfate
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
loss of approximately 50% of its activity within 2 h upon exposure to air
-
488148
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C or -80C, HEPES buffer with guanidine hydrochloride, alpha-cyclodextrin, ammonium sulfate, KCl and 20% (v/v) glycerol, several months, no significant loss of activity
0-5C, 0.1 M potassium phosphate buffer, pH 7.8, containing 20% glycerol and 1 mM dithiothreitol, about 50% loss of activity after 2 weeks
-
4-6C, enzyme in cell-free extract in presence of 20% glycerol, 65% loss of activity after 48 h
-
4C, buffer without stabilizing components, 72 h, complete loss of activity
4C, stable for at least 1 week
Saccharomycopsis lipolytica
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a Ni2+-nitrilotriacetic acid column, a gel filtration column, a HiLoad 26/60 phenyl-Sepharose column and a Resource Q anion exchange column are used
-
His-tagged enzyme
-
HisTrap column chromatography
-
Ni-NTA resin column chromatography
-
nickel affinity column chromatography
on a Talon Co2+ immobilized metal affinity chromatography column or a Zn2+ charged IMAC sepharose column
-
on Talon or Zn2+ charged IMAC sepharose resin
recombinant enzyme
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed at high levels in Escherichia coli
-
expressed in Escherichia coli B834 DE3 cells
-
expressed in Escherichia coli BL21(DE3) RIL star cells
-
expressed in Escherichia coli BL21(DE3) Rosetta 2 cells
expressed in Escherichia coli Rosetta (DE3) pLysS cells
-
expression in Escherichia coli
-
into the parallel expression vector pHIS2
-
overexpression in Penicillium chrysogenum using additional copies of lys1 with its own promoter or under the control of the pcbC promoter in either autonomously replicating or integrative vectors. Tranformants containing 3 to 32 additional copies of the lys1 gene are selected
-
three expression systems are designed, TtHCS without tag, with a C-terminal His6 tag and with a N-terminal His8 tag, the vectors pBluescript II SK+ and pET26b+ are used for expression in Escherichia coli cells
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
gene expression increases under conditions of lysine supplementation
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E155A
-
mutant enzyme exhibits 1000fold lower activity than wild-type enzyme. Activity of E155A can be partially rescued by formate
E155Q
-
mutant enzyme exhibits 1000fold lower activity than wild-type enzyme. The kcat for E155Q decreases at high pH, similar to the wild-type enzyme, but is pH independent at low pH
H309A
-
inactive mutant enzyme. Slight increase in activity is observed for H309A in the presence of 300 mM imidazole, which is still 1000fold lower than that of wild type
H309N
-
inactive mutant enzyme
Y320F
-
mutant enzyme loses 25fold activity compared to that of the wild-type enzyme
E167A
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
E167Q
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
E74A
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
E74Q
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
H103A
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
Q47A
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
R163A
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
R163K
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
R163Q
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
R43A
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
R43K
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
R43Q
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
S165A
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
T197A
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
T197S
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
T197V
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
Y332A
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
Y332F
-
mutant, reveals the contribution of this residue to substrate binding and catalysis
H72L
-
replacement of His72 by leucine makes HCS resistant to lysine inhibition, demonstrating the regulatory role of this conserved residue
additional information
-
activity and protein level of homocitrate synthase are dramatically reduced upon depletion of CuZn-superoxide dismutase. Overexpression of the lys4 gene increases homocitrate synthase activity and is sufficient to suppress the lysine requirement of CuZn-superoxide dismutase-deficient cells
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
the alteration of homocitrate synthase activity can be a useful strategy for improving sustained photobiological hydrogen production in cyanobacteria. Greater sustained H2 production and higher nitrogenase activities of the DELTAhupL DELTAnifV1 mutant culture grown under air
medicine
-
homocitrate synthase is a potential target for antifungal drugs
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