Deacetoxycephalosporin C hydroxylase of Streptomyces clavuligerus. Purification, characterization, bifunctionality, and evolutionary implication.
نویسندگان
چکیده
منابع مشابه
Deacetoxycephalosporin C synthetase and deacetoxycephalosporin C hydroxylase are two separate enzymes in Streptomyces clavuligerus.
pounds is accomplished by means of a biosynthetic pathway which closely parallels the corresponding pathway to cephalosporin C in Cephalosporium acremonium. Two of the enzymes involved in this pathway, deacetoxycephalosporin C (DAOC) synthetase and DAOC hydroxylase, carry out sequential reactions in which penicillin N undergoes ring expansion to DAOC and then DAOC is hydroxylated at the C3 meth...
متن کاملPurification and characterization of a 2-oxoglutarate-linked ATP-independent deacetoxycephalosporin C synthase of Streptomyces lactamdurans.
The 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-oxoglutara...
متن کاملDirected evolution of Streptomyces clavuligerus deacetoxycephalosporin C synthase for enhancement of penicillin G expansion.
The deacetoxycephalosporin C synthase from Streptomyces clavuligerus was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases ...
متن کاملEngineering Streptomyces clavuligerus deacetoxycephalosporin C synthase for optimal ring expansion activity toward penicillin G.
The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, ...
متن کاملPurification and properties of deacetoxycephalosporin C synthase from recombinant Escherichia coli and its comparison with the native enzyme purified from Streptomyces clavuligerus.
A putatively rate-limiting synthase (expandase) of Streptomyces clavuligerus was stabilized in vitro and purified 46-fold from cell-free extracts; a major enriched protein with a Mr of 35,000 was further purified by electrophoretic elution. Based on a 22-residue amino-terminal sequence of the protein, the synthase gene of S. clavuligerus was cloned and expressed in Escherichia coli (Kovacevic, ...
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ژورنال
عنوان ژورنال: Journal of Biological Chemistry
سال: 1991
ISSN: 0021-9258
DOI: 10.1016/s0021-9258(19)67759-8