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Literature summary for 2.3.1.165 extracted from

  • Moriguchi, T.; Kezuka, Y.; Nonaka, T.; Ebizuka, Y.; Fujii, I.
    Hidden function of catalytic domain in 6-methylsalicylic acid synthase for product release (2010), J. Biol. Chem., 285, 15637-15643.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.3.1.165

Cloned(Commentary)

Cloned (Comment) Organism
functional expression of ATX in Escherichia coli strain BL21(DE3)-CodonPlus RIPL, expression of N-terminally His6-tagged ATX and its thioester hydrolase domain, and the truncated mutant enzyme form, THID Aspergillus terreus

Protein Variants

Protein Variants Comment Organism
H972A site-directed mutagenesis, the reaction intermediate is bound to the thioester hydrolase domain of ATX formed in the presence of NADPH, and is released as 6-methylsalicylic acid by both the intact ATX and by truncated THID mutant in trans Aspergillus terreus
additional information construction of the THID mutant protein, a His-tagged 61 kDA 541-amino acid region containing thioester hydrolase domain and its downstream, THID shows catalytic activity to hydrolyze a model substrate 6-methylsalicylic acid-N-acetylcysteamine. The complementation of thioester domain-deficient mutant THm by THID protein in the recombinant Escherichia coli expression system Aspergillus terreus

Inhibitors

Inhibitors Comment Organism Structure
additional information no inhibition by phenylmethylsulfonyl fluoride, a serine protease-type thioesterase inhibitor at 1 mM Aspergillus terreus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
5.76
-
 6-Methylsalicylate release from enzyme/acyl-carrier-protein, recombinant His6-tagged THID mutant, pH 7.5, 25°C Aspergillus terreus
12.3
-
 6-Methylsalicylate release from enzyme/acyl-carrier-protein, recombinant His6-tagged ATX, pH 7.5, 25°C Aspergillus terreus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
acetyl-CoA + 3 malonyl-CoA + NADPH + H+ Aspergillus terreus
-
 6-methylsalicylate + 4 CoA + 3 CO2 + NADP+ + H2O
-
 ?

Organism

Organism UniProt Comment Textmining
Aspergillus terreus
-
 gene atx
-

Purification (Commentary)

Purification (Comment) Organism
recombinant N-terminally His6-tagged ATX and its thioester hydrolase domain, and the truncated mutant enzyme form, THID, from Escherichia coli strain BL21(DE3)-CodonPlus RIPL, by nickel affinity chromatography, and THID further by anion exchange chromatography, followed by gel filtration Aspergillus terreus

Reaction

Reaction Comment Organism Reaction ID
acetyl-CoA + 3 malonyl-CoA + NADPH + H+ = 6-methylsalicylate + 4 CoA + 3 CO2 + NADP+ + H2O 6-methylsalicylate synthesis proceeds without catalytic dehydration of the beta-hydroxy triketide intermediate by DH domain which is therefore not required for tetraketide formation. The subsequent aldol cyclization and aromatization of the tetraketide intermediate then form 6-methylsalicylate covalently bound to the ACP phosphopantetheine arm as the thioester. 6-methylsalicylate is released hydrolytically from ATX by the action of the domain hitherto called DH domain, i.e. the thioester hydrolase catalytic domain Aspergillus terreus
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
acetyl-CoA + 3 malonyl-CoA + NADPH + H+
-
 Aspergillus terreus 6-methylsalicylate + 4 CoA + 3 CO2 + NADP+ + H2O
-
 ?
acetyl-CoA + 3 malonyl-CoA + NADPH + H+ ATX by itself can synthesize tetraketide and release 6-methylsalicylate as the free acid without involvement of serine protease-type thioesterase, the product 6-methylsalicylate is retained on the acyl-carrier-protein until its release from ATX, product-releasing mechanism, overview Aspergillus terreus 6-methylsalicylate + 4 CoA + 3 CO2 + NADP+ + H2O
-
 ?
additional information the truncated enzyme, THID mutant, hydrolyzes the model substrate 6-methylsalicylic acid-N-acetylcysteamine Aspergillus terreus ?
-
 ?

Subunits

Subunits Comment Organism
More ATX does not have a conserved thioesterase GXSXG motif Aspergillus terreus
tetramer
-
 Aspergillus terreus

Synonyms

Synonyms Comment Organism
6-methylsalicylic acid synthase
-
 Aspergillus terreus
ATX
-
 Aspergillus terreus
MSAS
-
 Aspergillus terreus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Aspergillus terreus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0125
-
 6-Methylsalicylate release from enzyme/acyl-carrier-protein, recombinant His6-tagged THID mutant, pH 7.5, 25°C Aspergillus terreus
0.023
-
 6-Methylsalicylate release from enzyme/acyl-carrier-protein, recombinant His6-tagged ATX, pH 7.5, 25°C Aspergillus terreus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Aspergillus terreus

Cofactor

Cofactor Comment Organism Structure
NADPH
-
 Aspergillus terreus

General Information

General Information Comment Organism
evolution 6-methylsalicylic acid synthase belongs to the iterative type I polyketide synthases Aspergillus terreus
physiological function 6-methylsalicylic acid synthase from Aspergillus terreus is not involved in dehydration of the beta-hydroxytriketide intermediate tethered on the acyl carrier protein but catalyzes thioester hydrolysis to release the product from the acyl carrier protein, via its thioester hydrolase domain, a product-releasing domain that is located in the middle of a multidomain iterative type I polyketide synthase, ATX by itself can synthesize tetraketide and release 6-methylsalicylate as the free acid without involvement of serine protease-type thioesterase, product-releasing mechanism, overview Aspergillus terreus