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MetaCyc Enzyme: deacetoxycephalosporin C hydroxylase

Gene: cefF Accession Number: G-10201 (MetaCyc)

Species: Streptomyces clavuligerus

Summary:
The formation of deacetylcephalosporin C creates the second branch point in the β-lactam biosynthetic pathway leading to the biosynthesis of cephamycin C and cephalosporin C (see superpathway of penicillin, cephalosporin and cephamycin biosynthesis). 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 (Acremonium chrysogenum).

In S. clavuligerus two separate enzymes, deacetoxycephalosporin C synthase and deacetoxycephalosporin C hydroxylase, are used to catalyze two reactions that lead to the biosynthesis of cephamycin C. These two enzymes are encoded by the cefE 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]

The relative molecular mass of the deacetoxycephalosporin C hydroxylase polypeptide was determined to be 38 kDa by SDS-PAGE and 35 kDa by gel filtration chromatography. The deacetoxycephalosporin C hyroxylase from S. clavuligerus showed extensive sequence similarity to deacetoxycephalosporin C synthase from this organism, suggesting divergent evolution (in [Baker91].

Gene Citations: [Kovacevic91]

Molecular Weight of Polypeptide: 34.585 kD (from nucleotide sequence), 38.0 kD (experimental) [Baker91 ]

pI: 4.8 [Baker91]

Unification Links: Protein Model Portal:P42220 , SMR:P42220 , UniProt:P42220

Relationship Links: Entrez-Nucleotide:PART-OF:M63809 , InterPro:IN-FAMILY:IPR005123 , InterPro:IN-FAMILY:IPR026992 , InterPro:IN-FAMILY:IPR027443 , Pfam:IN-FAMILY:PF03171 , Pfam:IN-FAMILY:PF14226 , Prosite:IN-FAMILY:PS51471

Gene-Reaction Schematic: ?

Credits:
Created 20-Sep-2007 by Fulcher CA , SRI International


Enzymatic reaction of: deacetoxycephalosporin C hydroxylase

Synonyms: deacetylcephalosporin C synthase

EC Number: 1.14.11.26

deacetoxycephalosporin C + 2-oxoglutarate + oxygen <=> deacetylcephalosporin-C + succinate + CO2

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.

Alternative Substrates for deacetoxycephalosporin C: 3-exomethylenecephalosporin-C [Baker91 ]

In Pathways: superpathway of penicillin, cephalosporin and cephamycin biosynthesis , deacetylcephalosporin C biosynthesis

Summary:
Deacetoxycephalosporin C hydroxylase catalyzes the conversion of deacetoxycephalosporin C to deacetylcephalosporin C. In this reaction, the C-3 methyl group of deacetoxycephalosporin C is hydroxylated and oxidized to deacetylcephalosporin C. Like the reaction preceeding it, this is also an α-ketoglutarate (2-oxoglutarate) linked dioxygenase step. Reviewed in [Brakhage98] and [Thykaer03]

The enzyme also catalyzes the 3'-hydroxylation of the substrate analog 3-exomethylenecephalosporin-C, producing deacetylcephalosporin C at about one third the rate of the deacetoxycephalosporin C substrate. A slight ring expansion activity converting penicillin N to deacetoxycephalosporin C was also observed. Deacetylcephalosporin C and cephalosporin C did not serve as substrates. [Baker91]

The requirement for Fe2+ could not be replaced by Mg2+, Mn2+, Co2+, Ca2+, Cu2+, Ni2+, Zn2+, Na+, or K+. The stimulation by reducing agents was not observed using 2-mercaptoethanol.

Cofactors or Prosthetic Groups: Fe2+ [Baker91], 2-oxoglutarate [Baker91], oxygen [Baker91]

Activators (Unknown Mechanism): ATP [Baker91] , dithiothreitol [Baker91] , glutathione [Baker91]

Inhibitors (Competitive): penicillin N [Baker91]

Inhibitors (Unknown Mechanism): N-ethylmaleimide [Baker91] , iodoacetate [Baker91] , p-hydroxymercuribenzoate [Baker91] , 5,5'-dithio-bis-2-nitrobenzoate [Baker91] , o-phenanthroline [Baker91] , EDTA [Baker91] , Ni2+ [Baker91] , Mn2+ [Baker91] , Zn2+ [Baker91] , Co2+ [Baker91]

Kinetic Parameters:

Substrate
Km (μM)
Citations
2-oxoglutarate
10.0
[Baker91]
deacetoxycephalosporin C
59.0
[Baker91]

T(opt): 29 °C [Baker91]

pH(opt): 7.0-7.4 [Baker91]


References

Baker91: Baker BJ, Dotzlaf JE, Yeh WK (1991). "Deacetoxycephalosporin C hydroxylase of Streptomyces clavuligerus. Purification, characterization, bifunctionality, and evolutionary implication." J Biol Chem 266(8);5087-93. PMID: 2002049

Brakhage98: Brakhage AA (1998). "Molecular regulation of beta-lactam biosynthesis in filamentous fungi." Microbiol Mol Biol Rev 62(3);547-85. PMID: 9729600

Kovacevic91: Kovacevic S, Miller JR (1991). "Cloning and sequencing of the beta-lactam hydroxylase gene (cefF) from Streptomyces clavuligerus: gene duplication may have led to separate hydroxylase and expandase activities in the actinomycetes." J Bacteriol 173(1);398-400. PMID: 1987130

Thykaer03: Thykaer J, Nielsen J (2003). "Metabolic engineering of beta-lactam production." Metab Eng 5(1);56-69. PMID: 12749845


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by SRI International Pathway Tools version 18.5 on Mon Nov 24, 2014, BIOCYC13A.