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Literature summary for 3.5.1.11 extracted from

  • Varshney, N.K.; Ramasamy, S.; Brannigan, J.A.; Wilkinson, A.J.; Suresh, C.G.
    Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila (2013), Acta Crystallogr. Sect. F, 69, 925-929.
    View publication on PubMedView publication on EuropePMC

Application

Application Comment Organism
synthesis the enzyme KcPGA is quite resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin nucleus, which is the starting material for semisynthetic antibiotic production Kluyvera cryocrescens

Cloned(Commentary)

Cloned (Comment) Organism
gene pac, recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3) pLysS Kluyvera cryocrescens

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant enzyme, sitting drop vapour diffusion method, mixing of 300 nl of 45 mg/ml protein solution with 0.1 ml of precipitant solution containing 30% w/v PEG 4000, 50 mM sodium cacodylate pH 5.6, 0.5 M potassium thiocyanate, 3-4 days, mixing of 500 nl of protein and of precipitant solutions each results in crystals that grow in a week under a wide range of pH conditions using 50 mM sodium cacodylate buffer, method optimization, X-ray diffraction structure determination and analysis at 2.5-3.5 A resolution, molecular replacement method Kluyvera cryocrescens

Protein Variants

Protein Variants Comment Organism
S290G site-directed mutagenesis of the internal proteolytic cleavage site of the pro-enzyme Kluyvera cryocrescens

Localization

Localization Comment Organism GeneOntology No. Textmining

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
92000
-
1 * 92000, pro-enzyme, SDS-PAGE Kluyvera cryocrescens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
penicillin G + H2O Kluyvera cryocrescens
-
phenylacetate + 6-aminopenicillanate
-
?
penicillin G + H2O Kluyvera cryocrescens DMSZ 2660
-
phenylacetate + 6-aminopenicillanate
-
?

Organism

Organism UniProt Comment Textmining
Kluyvera cryocrescens P07941 gene pac
-
Kluyvera cryocrescens DMSZ 2660 P07941 gene pac
-

Posttranslational Modification

Posttranslational Modification Comment Organism
proteolytic modification the enzyme is synthesized as a single-chain inactive pro-enzyme, which upon autocatalytic processing becomes an active heterodimer of alpha and beta chains. Cleavage of the Thr289-Ser290 bond leads to the unveiling of the primary amine group of Serbeta1 (Ser290 of the precursor), creating the active centre in mature enzyme Kluyvera cryocrescens

Purification (Commentary)

Purification (Comment) Organism
recombinant N-terminally His-tagged wild-type and mutant enzymes to homogeneity from Escherichia coli strain BL21 (DE3) pLysS by nickel affinity chromatography and gel filtration Kluyvera cryocrescens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
penicillin G + H2O
-
Kluyvera cryocrescens phenylacetate + 6-aminopenicillanate
-
?
penicillin G + H2O
-
Kluyvera cryocrescens DMSZ 2660 phenylacetate + 6-aminopenicillanate
-
?

Subunits

Subunits Comment Organism
heterodimer
-
Kluyvera cryocrescens
monomer 1 * 92000, pro-enzyme, SDS-PAGE Kluyvera cryocrescens

Synonyms

Synonyms Comment Organism
penicillin G acylase
-
Kluyvera cryocrescens
PGA
-
Kluyvera cryocrescens