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Literature summary extracted from

  • Sagong, H.Y.; Hong, J.; Kim, K.J.
    Crystal structure and biochemical characterization of O-acetylhomoserine acetyltransferase from Mycobacterium smegmatisATCC 19420 (2019), Biochem. Biophys. Res. Commun., 517, 399-406 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.3.1.31 gene metX_1, sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)-T1 Mycolicibacterium smegmatis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.3.1.31 purified recombinant His-tagged enzyme in apo-form and in complex with either CoA or homoserine, hanging drop vapor diffusion method, mixing of 0.001 ml of protein solution containing 40 mg/ml protein, and 40 mM Tris-HCl, pH 8.0, with 0.001 ml of reservoir solution containing 34% w/v PEG 400, 0.1 M sodium acetate/acetic acid, pH 5.5, and 0.2 M calcium acetate hydrate, and additionally 10 mM ligand for the complex crystals, equilibrating against 0.5 ml of reservoir solution, 20°C, X-ray diffraction structure determination and analysis at 1.55 A resolution Mycolicibacterium smegmatis

Protein Variants

EC Number Protein Variants Comment Organism
2.3.1.31 D315A site-directed mutagenesis, the mutant shows an almost complete loss of activity Mycolicibacterium smegmatis
2.3.1.31 H345A site-directed mutagenesis, the mutant shows an almost complete loss of activity Mycolicibacterium smegmatis
2.3.1.31 K267A site-directed mutagenesis, the mutant shows a slight loss in activity Mycolicibacterium smegmatis
2.3.1.31 L55A site-directed mutagenesis, the mutant shows a slight loss in activity Mycolicibacterium smegmatis
2.3.1.31 Q244A site-directed mutagenesis, the mutant shows a slight loss in activity Mycolicibacterium smegmatis
2.3.1.31 R238A site-directed mutagenesis, the mutant shows a slight loss in activity Mycolicibacterium smegmatis
2.3.1.31 S152A site-directed mutagenesis, the mutant shows an almost complete loss of activity Mycolicibacterium smegmatis
2.3.1.31 S259A site-directed mutagenesis, the mutant shows a slight loss in activity Mycolicibacterium smegmatis
2.3.1.31 T56A site-directed mutagenesis, the mutant shows a slight loss in activity Mycolicibacterium smegmatis
2.3.1.31 Y263A site-directed mutagenesis, the mutant shows a slight loss in activity Mycolicibacterium smegmatis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.3.1.31 additional information
-
 additional information kinetic analysis Mycolicibacterium smegmatis
2.3.1.31 0.06
-
 L-homoserine recombinant His-tagged enzyme, pH 8.0, 22°C Mycolicibacterium smegmatis
2.3.1.31 0.158
-
 acetyl-CoA recombinant His-tagged enzyme, pH 8.0, 22°C Mycolicibacterium smegmatis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.3.1.31 acetyl-CoA + L-homoserine Mycolicibacterium smegmatis
-
 CoA + O-acetyl-L-homoserine
-
 ?
2.3.1.31 acetyl-CoA + L-homoserine Mycolicibacterium smegmatis ATCC 19420
-
 CoA + O-acetyl-L-homoserine
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.3.1.31 Mycolicibacterium smegmatis A0A8B4QGM8 i.e. Mycolicibacterium smegmatis
-
2.3.1.31 Mycolicibacterium smegmatis ATCC 19420 A0A8B4QGM8 i.e. Mycolicibacterium smegmatis
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.3.1.31 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3)-T1 by nickel affinity chromatography and two different steps of gel filtration Mycolicibacterium smegmatis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.3.1.31 acetyl-CoA + L-homoserine
-
 Mycolicibacterium smegmatis CoA + O-acetyl-L-homoserine
-
 ?
2.3.1.31 acetyl-CoA + L-homoserine
-
 Mycolicibacterium smegmatis ATCC 19420 CoA + O-acetyl-L-homoserine
-
 ?

Subunits

EC Number Subunits Comment Organism
2.3.1.31 dimer MsHAT functions as a dimer. For dimerization, the alpha3 and alpha4 of the lid domain from each subunit form an antiparallel four-helix bundle with the hydrophobic core and the two helices from each subunit surround the dimerization center. In addition, the alpha5 and alpha5-alpha6 connecting loop extend to the other monomer and interact with residues from the other molecule through hydrogen bonds and salt bridges. The conformation of the alpha5 to alpha6 region might influence the shape of the dimer. The active site entrance shows an open or closed conformation and might determine the substrate binding affinity of HAT enzymes Mycolicibacterium smegmatis
2.3.1.31 More the monomeric structure of MsHAT consists of two distinctive domains: a core domain (residues Met1-Ala176 and His287-Arg368) and a lid domain (Val177-Ser286). The core domain shows alpha/beta hydrolase fold and contains an eight-stranded parallel beta-sheet with four alpha-helices on one side and one alpha-helix on the opposite site. The lid domain is inserted between beta8 and alpha8 and is composed of five alpha-helices, and it mainly contributes to dimerization of MsHAT. MsHAT functions as a dimer Mycolicibacterium smegmatis

Synonyms

EC Number Synonyms Comment Organism
2.3.1.31 HAT
-
 Mycolicibacterium smegmatis
2.3.1.31 metX_1
-
 Mycolicibacterium smegmatis
2.3.1.31 MsHAT
-
 Mycolicibacterium smegmatis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.3.1.31 22
-
 assay at room temperature Mycolicibacterium smegmatis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.3.1.31 2.918
-
 L-homoserine recombinant His-tagged enzyme, pH 8.0, 22°C Mycolicibacterium smegmatis
2.3.1.31 3.074
-
 acetyl-CoA recombinant His-tagged enzyme, pH 8.0, 22°C Mycolicibacterium smegmatis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.3.1.31 8
-
 assay at Mycolicibacterium smegmatis

Cofactor

EC Number Cofactor Comment Organism Structure
2.3.1.31 acetyl-CoA
-
 Mycolicibacterium smegmatis

General Information

EC Number General Information Comment Organism
2.3.1.31 additional information substrate binding mode and molecular mechanism of MsHAT, detailed overview. Enzyme structure comparisons. The active site entrance shows an open or closed conformation and might determine the substrate binding affinity of HAT enzymes. The conserved Ser152, His345, and Asp315 residues form a catalytic triad, and they act as a covalent nucleophile, a general base, and an electron donor, respectively. Arg222, Asp346, Tyr229, and Tyr260 residues are mainly involved in the binding of homoserine or acetyl-CoA and other residues are not crucial for the stabilization of its substrates Mycolicibacterium smegmatis
2.3.1.31 physiological function enzyme MsHAT catalyzes the transfer of acetyl-group from acetyl-CoA to homoserine Mycolicibacterium smegmatis