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MetaCyc Enzyme: deacetoxycephalosporin C synthase / deacetoxycephalosporin C hydroxylase

Gene: CEFEF Accession Number: G-10199 (MetaCyc)

Synonyms: deacetoxycephalosporin/deacetylcephalosporin C synthase, deacetoxycephalosporin C synthetase/hydroxylase

Species: Acremonium chrysogenum

Summary:
Penicillin N is a precursor of the β-lactam antibiotics cephalosporin C and cephamycin C. Cephalosporin C and other cephalosporins are biosynthesized by fungi such as Acremonium chrysogenum (Acremonium chrysogenum). Cephamycin C and other cephamycins (7-methoxycephalosporins) are biosynthesized by bacteria such as Streptomyces clavuligerus.

Deacetoxycephalosporin C synthase (expandase) catalyzes the conversion of penicillin N to deacetoxycephalosporin C. In A. chrysogenum this is a bifunctional enzyme (deacetoxycephalosporin C synthase/deacetoxycephalosporin C hydroxylase) that also catalyzes the next reaction in the cephalosporin C biosynthetic pathway, deacetoxycephalosporin C hydroxylase (synonym deacetylcephalosporin C synthase), converting deacetoxycephalosporin C to deacetylcephalosporin C. This bifunctional enzyme is encoded by the cefEF gene.

In contrast, S. clavuligerus uses two separate enzymes, deacetoxycephalosporin C synthase and deacetoxycephalosporin C hydroxylase, to catalyze these reactions that lead to the biosynthesis of cephamycin C in this organism. These two enzymes are encoded by the cef E and cefF genes, respectively.

Deacetoxycephalosporin C hydroxylase catalyzes the conversion of deacetoxycephalosporin C to deacetylcephalosporin C. The formation of deacetylcephalosporin C creates the second branch point in the β-lactam biosynthetic pathway leading to the biosynthesis of cephalosporin C and cephamycin C (see superpathway of penicillin, cephalosporin and cephamycin biosynthesis). Reviewed in [Brakhage98] and [Thykaer03]

The A. chrysogenum enzyme is an iron(II) and 2-oxoglutarate-dependent oxygenase. Its reaction and substrate selectivity have been studied using structural models and site-directed mutagenesis [Lloyd04a]. The recombinant enzyme has been overexpressed as granules (inclusion bodies) in Escherichia coli, purified, and refolded to an active state [Ghag96].

The relative molecular mass of the native, purified enzyme was estimated to be 43 kDa by gel filtration chromatography and 41 kDa by SDS-PAGE [Dotzlaf87].

Molecular Weight of Polypeptide: 36.479 kD (from nucleotide sequence), 41.0 kD (experimental) [Dotzlaf87 ]

pI: 6.3 [Dotzlaf87]

Unification Links: Protein Model Portal:P11935 , UniProt:P11935

Relationship Links: Entrez-Nucleotide:PART-OF:AJ404737 , InterPro:IN-FAMILY:IPR002057 , InterPro:IN-FAMILY:IPR005123 , InterPro:IN-FAMILY:IPR026992 , InterPro:IN-FAMILY:IPR027443 , Pfam:IN-FAMILY:PF03171 , Pfam:IN-FAMILY:PF14226 , Prosite:IN-FAMILY:PS00185 , Prosite:IN-FAMILY:PS00186 , 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.

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

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

Assays for hydroxylase activity using bioassay and thin-layer chromatograhy, or high-performance liquid chromatography have been described [Baldwin87, Dotzlaf87]. The requirement for Fe2+ could not be replaced by Mg2+, Mn2+, Co2+, Ca2+, Cu2+, Ni2+, Zn2+, Na+, or K+. Unlike other sulfhydryl reagents, iodoacetate and N-ethylmaleimide did not inhibit hydroxylase activity. [Dotzlaf87].

Cofactors or Prosthetic Groups: 2-oxoglutarate [Dotzlaf87], Fe2+ [Dotzlaf87], oxygen [Dotzlaf87]

Activators (Unknown Mechanism): dithiothreitol [Dotzlaf87] , L-ascorbate [Dotzlaf87] , ATP [Dotzlaf87]

Inhibitors (Unknown Mechanism): p-hydroxymercuribenzoate [Dotzlaf87] , 5,5'-dithio-bis-2-nitrobenzoate [Dotzlaf87] , EDTA [Dotzlaf87] , o-phenanthroline [Dotzlaf87] , Mn2+ [Dotzlaf87] , Cu2+ [Dotzlaf87] , Zn2+ [Dotzlaf87] , Co2+ [Dotzlaf87]

Kinetic Parameters:

Substrate
Km (μM)
Citations
2-oxoglutarate
22.0
[Dotzlaf87]
deacetoxycephalosporin C
20.0
[Dotzlaf87]

T(opt): 36-38 °C [Dotzlaf87]

pH(opt): 7.3 [Dotzlaf87]


Enzymatic reaction of: deacetoxycephalosporin C synthase

Synonyms: deacetoxycephalosporin C synthetase

EC Number: 1.14.20.1

2-oxoglutarate + penicillin N + oxygen <=> CO2 + succinate + deacetoxycephalosporin C + H2O

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.

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

Summary:
This reaction involves ring expansion (expandase). 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]

Assays for synthase (expandase) activity using bioassay, or high-performance liquid chromatography have been described [Baldwin87, Dotzlaf87]. The enzyme acted on penicillin N as substrate, but not isopenicillin N, penicillin G, penicillin V, ampicillin, or 6-aminopenicillanate. The requirement for Fe2+ could not be replaced by Mg2+, Mn2+, Co2+, Ca2+, Cu2+, Ni2+, Zn2+, Na+, or K+. Unlike other sulfhydryl reagents, iodoacetate did not inhibit expandase activity. [Dotzlaf87].

Cofactors or Prosthetic Groups: oxygen [Dotzlaf87], 2-oxoglutarate [Baldwin87, Dotzlaf87], Fe2+ [Dotzlaf87]

Activators (Unknown Mechanism): L-ascorbate [Dotzlaf87, Baldwin87] , dithiothreitol [Dotzlaf87, Baldwin87] , ATP [Dotzlaf87]

Inhibitors (Unknown Mechanism): 4,7-diphenyl-1,10-phenanthroline [Baldwin87] , 5,5'-dithio-bis-2-nitrobenzoate [Dotzlaf87] , N-ethylmaleimide [Dotzlaf87, Baldwin87] , o-phenanthroline [Dotzlaf87] , p-hydroxymercuribenzoate [Dotzlaf87] , Mn2+ [Dotzlaf87] , EDTA [Dotzlaf87] , Zn2+ [Dotzlaf87] , Co2+ [Dotzlaf87]

Kinetic Parameters:

Substrate
Km (μM)
Citations
2-oxoglutarate
22.0
[Dotzlaf87]
penicillin N
29.0
[Dotzlaf87]

T(opt): 26-34 °C [Dotzlaf87]

pH(opt): 7.5-7.8 [Dotzlaf87]


References

Baldwin87: Baldwin JE, Adlington RM, Coates JB, Crabbe MJ, Crouch NP, Keeping JW, Knight GC, Schofield CJ, Ting HH, Vallejo CA (1987). "Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities." Biochem J 245(3);831-41. PMID: 3663194

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

Dotzlaf87: Dotzlaf JE, Yeh WK (1987). "Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium." J Bacteriol 169(4);1611-8. PMID: 3558321

Ghag96: Ghag SK, Brems DN, Hassell TC, Yeh WK (1996). "Refolding and purification of Cephalosporium acremonium deacetoxycephalosporin C synthetase/hydroxylase from granules of recombinant Escherichia coli." Biotechnol Appl Biochem 24 ( Pt 2);109-19. PMID: 8865604

Lloyd04a: Lloyd MD, Lipscomb SJ, Hewitson KS, Hensgens CM, Baldwin JE, Schofield CJ (2004). "Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase." J Biol Chem 279(15);15420-6. PMID: 14734549

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, biocyc14.