|Gene:||cefE||Accession Number: G-10200 (MetaCyc)|
Species: Streptomyces clavuligerus
Penicillin N is a precursor of the β-lactam antibiotics cephamycin C and cephalosporin C. Cephamycin C and other cephamycins (7-methoxycephalosporins) are biosynthesized by bacteria such as Streptomyces clavuligerus. Cephalosporin C and other cephalosporins are biosynthesized by fungi such as Acremonium chrysogenum (previously known as Cephalosporium acremonium). See superpathway of penicillin, cephalosporin and cephamycin biosynthesis.
In S. clavuligerus two separate enzymes, deacetoxycephalosporin C synthase and deacetoxycephalosporin C hydroxylase, are used to catalyze two sequential reactions that lead to the biosynthesis of cephamycin C. These two enzymes are encoded by the cef E and cefF genes, respectively. In contrast, A. chrysogenum uses a bifunctional enzyme deacetoxycephalosporin C synthase / deacetoxycephalosporin C hydroxylase to catalyze these two reactions that lead to the biosynthesis of cephalosporin C in this organism. This bifunctional enzyme is encoded by the cefEF gene. Reviewed in [Brakhage98] and [Thykaer03]
Both native and recombinant S. clavuligerus enzymes were found to have similar biochemical properties. Data given here are for the recombinant enzyme [Dotzlaf89].
Molecular Weight of Polypeptide: 34.556 kD (from nucleotide sequence)
Relationship Links: Entrez-Nucleotide:RELATED-TO:M32324 , InterPro:IN-FAMILY:IPR002057 , InterPro:IN-FAMILY:IPR005123 , InterPro:IN-FAMILY:IPR026992 , InterPro:IN-FAMILY:IPR027443 , PDB:Structure:1DCS , PDB:Structure:1E5H , PDB:Structure:1E5I , PDB:Structure:1HJF , PDB:Structure:1HJG , PDB:Structure:1RXF , PDB:Structure:1RXG , PDB:Structure:1UNB , PDB:Structure:1UO9 , PDB:Structure:1UOB , PDB:Structure:1UOF , PDB:Structure:1UOG , PDB:Structure:1W28 , PDB:Structure:1W2A , PDB:Structure:1W2N , PDB:Structure:1W2O , PDB:Structure:2JB8 , Pfam:IN-FAMILY:PF03171 , Pfam:IN-FAMILY:PF14226 , Prosite:IN-FAMILY:PS00185 , Prosite:IN-FAMILY:PS00186 , Prosite:IN-FAMILY:PS51471
Enzymatic reaction of: deacetoxycephalosporin C synthase
EC Number: 18.104.22.168
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.
The reaction is physiologically favored in the direction shown.
Deacetoxycephalosporin C synthase (expandase) catalyzes the conversion of penicillin N to deacetoxycephalosporin C. This reaction involves ring expansion. The five-membered penam thiazolidine ring is oxidatively opened and after closure, the six-membered dihydrothiazine ring is formed. This ring is characteristic of ceph-3-ems. The enzyme is an α-ketoglutarate (2-oxoglutarate) linked dioxygenase. Reviewed in [Brakhage98] and [Thykaer03]
Neither the native, nor the recombinant enzyme was stimulated by ATP, in contrast to the Acremonium chrysogenum enzyme. The recombinant enzyme also catalyzed a novel hydroxylation of 3-exomethylenecephalosporin-C to deacetylcephalosporin C similar to the Acremonium chrysogenum enzyme, but did not catalyze the hydroxylation of deacetoxycephalosporin C. 3-exomethylenecephalosporin-C competitively inhibited the enzyme with respect to penicillin N [Dotzlaf89].
Inhibitors (Unknown Mechanism): penicillin G [Dotzlaf89] , penicillin V [Dotzlaf89] , 5,5'-dithio-bis-2-nitrobenzoate [Dotzlaf89] , N-ethylmaleimide [Dotzlaf89] , o-phenanthroline [Dotzlaf89] , p-hydroxymercuribenzoate [Dotzlaf89] , EDTA [Dotzlaf89]
T(opt): 36 °C [Dotzlaf89]
pH(opt): 7.0 [Dotzlaf89]
Dotzlaf89: Dotzlaf JE, Yeh WK (1989). "Purification and properties of deacetoxycephalosporin C synthase from recombinant Escherichia coli and its comparison with the native enzyme purified from Streptomyces clavuligerus." J Biol Chem 264(17);10219-27. PMID: 2656705
Kovacevic89: Kovacevic S, Weigel BJ, Tobin MB, Ingolia TD, Miller JR (1989). "Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase." J Bacteriol 171(2);754-60. PMID: 2644235
Kovacevic90: Kovacevic S, Tobin MB, Miller JR (1990). "The beta-lactam biosynthesis genes for isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus." J Bacteriol 172(7);3952-8. PMID: 1694525
©2014 SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025-3493