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Literature summary for 2.3.1.194 extracted from

  • Okamura, E.; Tomita, T.; Sawa, R.; Nishiyama, M.; Kuzuyama, T.
    Unprecedented acetoacetyl-coenzyme A synthesizing enzyme of the thiolase superfamily involved in the mevalonate pathway (2010), Proc. Natl. Acad. Sci. USA, 107, 11265-11270.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.3.1.194

Application

Application Comment Organism
synthesis nphT7 can be used to significantly increase the concentration of acetoacetyl-CoA in cells, eventually leading to the production of useful terpenoids and poly-3-hydroxybutyrate Streptomyces sp.

Cloned(Commentary)

Cloned (Comment) Organism
gene nphT7, organized in the mevalonate pathway gene cluster, functional expression in Streptomyces albus, overexpression of N-terminally His8-tagged enzyme in Escherichia coli Streptomyces sp.
overexpressed in Escherichia coli as an N-terminal His8-tagged protein Streptomyces sp.

Protein Variants

Protein Variants Comment Organism
C115A consumption of malonyl-CoA and an increase in acetyl-CoA. No formation of CoA or acetoacetyl-CoA. C115A mutant enzyme yields acetyl-CoA via its malonyl-CoA decarboxylation activity, presumably by the His256 and Asn286 residues, while it has lost its condensation activity Streptomyces sp.
C115A site-directed mutagenesis, the mutant cleaves malonyl-CoA into acetyl-CoA, but does not form CoA or acetoacetyl-CoA. C115A mutant enzyme yields acetyl-CoA via its malonyl-CoA decarboxylation activity, presumably by the His256 and Asn286 residues, while it has lost its condensation activity Streptomyces sp.
H256A can not ve obtained as a soluble protein Streptomyces sp.
H256A site-directed mutagenesis, the mutant enzyme exhibits acetoacetyl-CoA synthesizing activity, but its specific activity is approximately 40fold lower than that of wild-type NphT7 Streptomyces sp.
N286A this mutant enzyme exhibits detectable acetoacetyl-CoA synthesizing activity, but its specific is approximately 40fold lower than that of wild-type NphT7 Streptomyces sp.
N286A site-directed mutagenesis, the mutant cannot be produced recombionantly as a soluble protein Streptomyces sp.

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information steady-state kinetics Streptomyces sp.
0.028
-
 malonyl-CoA pH 8.0, 25°C Streptomyces sp.
0.028
-
 malonyl-CoA pH 8.0, 30°C, reaction with acetyl-CoA and malonyl-CoA Streptomyces sp.
0.028
-
 malonyl-CoA pH 8.0, 30°C, reaction with malonyl-CoA, without acetyl-CoA Streptomyces sp.
0.068
-
 acetyl-CoA pH 8.0, 25°C Streptomyces sp.
0.068
-
 acetyl-CoA pH 8.0, 30°C, reaction with acetyl-CoA and malonyl-CoA Streptomyces sp.

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
37000
-
 2 * 37000, SDS-PAGE Streptomyces sp.
37000
-
 2 * 37000, recombinant N-terminally His8-tagged enzyme, SDS-PAGE Streptomyces sp.
63000
-
 gel filtration Streptomyces sp.
63000
-
 recombinant N-terminally His8-tagged enzyme, gel filtration Streptomyces sp.

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
acetyl-CoA + malonyl-CoA Streptomyces sp. the acetoacetyl-CoA synthesizing enzyme increases the acetoacetyl-CoA supply for mevalonate production acetoacetyl-CoA + CoA + CO2
-
 ?
acetyl-CoA + malonyl-CoA Streptomyces sp.
-
 CoA + acetoacetyl-CoA + CO2
-
 ir

Organism

Organism UniProt Comment Textmining
Streptomyces sp.
-
-
-
Streptomyces sp.
-
 soil-isolated strain, gene nphT7
-

Purification (Commentary)

Purification (Comment) Organism
-
 Streptomyces sp.

Reaction

Reaction Comment Organism Reaction ID
acetyl-CoA + malonyl-CoA = acetoacetyl-CoA + CoA + CO2 the highly conserved catalytic triad is formed by residues Cys115, His256, and Asn286 in NphT7, reaction mechanism, overview Streptomyces sp.
a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.23
-
 recombinant mutant H256A, pH 8.0, 25°C Streptomyces sp.
8.9
-
 recombinant His8-tagged wild-type enzyme, pH 8.0, 25°C Streptomyces sp.

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 malonyl-CoA
-
 Streptomyces sp. acetoacetyl-CoA + CoA + 2 CO2
-
 ?
acetyl-CoA + malonyl-CoA the acetoacetyl-CoA synthesizing enzyme increases the acetoacetyl-CoA supply for mevalonate production Streptomyces sp. acetoacetyl-CoA + CoA + CO2
-
 ?
acetyl-CoA + malonyl-CoA Cys115 in NphT7 functions as a key catalytic residue for the condensation reaction. His256 functions as a catalytic residue for the decarboxylation of the extender substrate malonyl-CoA. No acetoacetyl-CoA synthesizing activity is detected through the condensation of two molecules of acetyl-CoA Streptomyces sp. acetoacetyl-CoA + CoA + CO2
-
 ?
acetyl-CoA + malonyl-CoA
-
 Streptomyces sp. CoA + acetoacetyl-CoA + CO2
-
 ir
additional information the recombinant enzyme catalyzes a single condensation of acetyl-CoA and malonyl-CoA to give acetoacetyl-CoA and CoA. Replacement of malonyl-CoA with malonyl-(acyl carrier protein) results in loss of the condensation activity. No acetoacetyl-CoA synthesizing activity detectable through the condensation of two molecules of acetyl-CoA Streptomyces sp. ?
-
 ?

Subunits

Subunits Comment Organism
dimer 2 * 37000, SDS-PAGE Streptomyces sp.
dimer 2 * 37000, recombinant N-terminally His8-tagged enzyme, SDS-PAGE Streptomyces sp.

Synonyms

Synonyms Comment Organism
acetoacetyl-coenzyme A synthesizing enzyme
-
 Streptomyces sp.
More the enzyme belongs to the thiolase superfamily of enzymes Streptomyces sp.
NphT7
-
 Streptomyces sp.
thiolase superfamily acetoacetyl-CoA synthase
-
 Streptomyces sp.

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Streptomyces sp.
30
-
 assay at Streptomyces sp.

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Streptomyces sp.

General Information

General Information Comment Organism
metabolism the acetoacetyl-coenzyme A synthesizing enzyme of the thiolase superfamily is involved in the mevalonate pathway. Acetoacetyl-CoA is the precursor of 3-hydroxy-3-methylglutaryl-CoA in the mevalonate pathway, which is essential for terpenoid backbone biosynthesis. Acetoacetyl-CoA is also the precursor of poly-3-hydroxybutyrate, a polymer belonging to the polyester class produced by microorganisms Streptomyces sp.