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Title

Purification and characterization of a 2-oxoglutarate-linked ATP-independent deacetoxycephalosporin C synthase of streptomyces lactamdurans

AuthorsCortés, Jesús ; Martín Martín, Juan Fco.; Castro González, José Mª; Laiz Trobajo, L. ; Liras Padín, P.
Issue DateNov-1987
PublisherSociety for General Microbiology
CitationJournal of General Microbiology 133: 3165-3174 (1987)
AbstractThe deacetoxycephalosporin C (DAOC) synthase (expandase) of Streptomyces lactamdurans was highly purified, as shown by SDS-PAGE and isoelectric focusing. The enzyme catalysed the oxidative ring expansion that converts penicillin N into DAOC. The enzyme was very unstable but could be partially stabilized in 25 mM-Tris/HCl, pH 9.0, in the presence of DTT (0.1 mM). The enzyme required 2-oxoglutarate, oxygen and Fe2+,but did not need ATP, ascorbic acid, Mg2+or K+.The optimum temperature was between 25 and 30 "C. The DAOC synthase showed a high specificityfor the penicillin substrate. Only penicillin N but not isopenicillin N, penicillin G or 6-aminopenicillanic acid served as substrates. 2-Oxoglutarate analogues were not used as substrates although 2-oxobutyrate and 3-oxoadipate inhibited the enzyme by 100% and 56% respectively. The enzyme was strongly inhibited by Cu2+,Co2+and Zn2+.The apparent K, values for penicillin N, 2-oxoglutarate and Fe2+were 52 PM, 3 PM and 71 PM respectively. The enzyme was a monomer with a molecular mass of 27000 Da & 1000.
Description10 pages, 5 figures, 3 tables, 21 references.
Publisher version (URL)http://dx.doi.org/10.1099/00221287-133-11-3165
URIhttp://hdl.handle.net/10261/63768
DOI10.1099/00221287-133-11-3165
ISSN1350-0872
E-ISSN1465-2080
Appears in Collections:(IRNAS) Artículos
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