Partial purification and catalytic properties of a bifunctional enzyme in the biosynthetic pathway of .BETA.-lactams in Cephalosporium acremonium.

Scheidegger, Alfred; Küenzi, Martin; Nüesch, J.

doi:10.7164/antibiotics.37.522

Cited by 98 publications

(62 citation statements)

References 21 publications

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“…and expressed in Escherichia coli (6a, 26). On the other hand, lability of the expandase has prevented significant purification of the enzyme (14,16,27). Recently, the expandase and DAOC hydroxylase in extracts of the procaryote Streptomyces clavuligerus were separated (11).…”

mentioning

confidence: 99%

Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium

Dotzlaf¹,

Yeh²

1987

J Bacteriol

121

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Deacetoxycephalosporin C synthetase (expandase), which catalyzes ring expansion of penicillin N to deacetoxycephalosporin C (DAOC), has been stabilized in vitro and purified to near homogeneity from the industrially important fungus Cephalosporium acremonium. Throughout the purification, the expandase activity remained physically associated with and in a constant ratio of 7:1 to DAOC hydroxylase activity. The latter activity mediates hydroxylation of DAOC to deacetylcephalosporin C (DAC). The copurified expandase/hydroxylase appeared to be monomeric, with a molecular weight of 41,000 +/- 2,000 and an isoelectric point of 6.3 +/- 0.3. Both catalytic activities required alpha-ketoglutarate, Fe2+, and O2 and were stimulated by ascorbate, dithiothreitol, and ATP. The Fe2+ requirement was specific, and sulfhydryl groups in the purified protein were apparently essential for both ring expansion and hydroxylation. The kinetics and stoichiometry of DAOC/DAC formation from the expandase/hydroxylase-catalyzed reactions suggested that ring expansion of penicillin N preceded hydroxylation of DAOC.

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mentioning

confidence: 99%

Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium

Dotzlaf¹,

Yeh²

1987

J Bacteriol

121

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“…Expression of the cloned gene in E. coli demonstrated both DAOCS and DACS activities in the bacterium, thereby unequivocally establishing the bifunctional nature of the enzyme. Previously, bifunctionality had been suggested by the inability to separate the expandase and hydroxylase activities during purification of the enzyme from C. acremonium extracts (6,22). DAOCS activity may be the rate-limiting step in the production of cephalosporin C in certain strains of C. acremonium (22).…”

mentioning

confidence: 99%

“…Previously, bifunctionality had been suggested by the inability to separate the expandase and hydroxylase activities during purification of the enzyme from C. acremonium extracts (6,22). DAOCS activity may be the rate-limiting step in the production of cephalosporin C in certain strains of C. acremonium (22). Indeed, a C. acremonium strain that contains an extra cloned copy of the cefEF gene produces less penicillin N and more cephalosporin C than does its nonrecombinant parent; large-scale experiments with the improved strain are pending (8) (3,17,25; Ingolia and Queener, in press).…”

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confidence: 99%

Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase

et al. 1989

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Biosynthesis of cephalosporin antibiotics involves an expansion of the five-membered thiazolidine ring of penicillin N to the six-membered dihydrothiazine ring of deacetoxycephalosporin C by a deacetoxycephalosporin C synthetase (DAOCS) enzyme activity. Hydroxylation of deacetoxycephalosporin C to form deacetylcephalosporin C by a deacetylcephalosporin C synthetase (DACS) activity is the next step in biosynthesis of cephalosporins. In Cephalosporium acremonium, both of these catalytic activities are exhibited by a bifunctional enzyme, DAOCS-DACS, encoded by a single gene, cefEF. In Streptomyces clavuligerus, separable enzymes, DAOCS (expandase) and DACS (hydroxylase), catalyze these respective reactions. We have cloned, sequenced, and expressed in E. coli an S. clavuligerus gene, designated cefE, which encodes DAOCS but not DACS. The deduced amino acid sequence of DAOCS from S. clavuligerus (calculated Mr of 34,519) shows marked similarity (approximately 57%) to the deduced sequence of DAOCS-DACS from C. acremonium; however, the latter sequence is longer by 21 amino acid residues.

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“…The IPN is then isomerized to the d -configuration by two linked genes, cefD1-cefD2 , giving rise to penicillin N (PenN) (89,121) , which is later converted to deacetoxy cephalosporin C (DAOC) by a ring expansion step catalyzed by the DAOC synthase (encoded by the cefEF gene). The latter enzyme is also able to oxidize the methyl group at carbon 3 ′ of DAOC to give deacetylcephalosporin C (DAC) (122,123) . Both activities are located in a single polypeptide that is encoded by the cefEF gene (124) .…”

Section: Role Of Two Peroxisomal Membrane Transporters In Cephalospormentioning

confidence: 99%

Transport of substrates into peroxisomes: the paradigm of β-lactam biosynthetic intermediates

Martı́n

Garcı́a-Estrada

Ullán

2013

BioMolecular Concepts

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Peroxisomes are ubiquitous organelles that enclose catalases, fatty acid-oxidizing enzymes, and a variety of proteins involved in different cellular processes. Interestingly, the late enzymes involved in penicillin biosynthesis, and the isopenicillin N epimerization enzymes involved in cephalosporin biosynthesis are located inside peroxisomes in the producer fungi Penicillium chrysogenum and Acremonium chrysogenum. Peroxisome proteins are targeted to those organelles by peroxisomal targeting signals located at the C-terminus (PTS1) or near the N-terminal end (PTS2) of those proteins. Peroxisomal membrane proteins (PMPs) are largely recruited by the interaction with specific sequences in the Pex19 protein. The compartmentalization into peroxisomes of several steps of the biosynthesis of penicillin, cephalosporin, and other secondary metabolites raises the question of how the precursors and/or intermediates of the biosynthesis of β -lactam antibiotics are transported into peroxisomes and the mechanisms of secretion of the final products (penicillin or cephalosporin) from peroxisomes to the extracellular medium. Recent advances in peroxisome proteomics, immunoelectron microscopy, and fluorescence labeling have shown that the transport of these intermediates is mediated by membrane proteins of the major facilitator superfamily class (drug/H + antiporters) containing 12 transmembrane-spanning domains (TMS). In some cases, the transport of the substrates (e.g., fatty acids) or intermediates may be mediated by ATP-binding cassette (ABC) transporters. Knowledge on the transport and secretion mechanisms is of paramount importance to understand the complex mechanisms of cell differentiation and their crosstalk with the biosynthesis of different secondary metabolites that act as biochemical signals between the producer cells and also as communication signals with competing microorganisms (e.g., antimicrobial agents or plant elicitors).

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Partial purification and catalytic properties of a bifunctional enzyme in the biosynthetic pathway of .BETA.-lactams in Cephalosporium acremonium.

Cited by 98 publications

References 21 publications

Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium

Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium

Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase

Transport of substrates into peroxisomes: the paradigm of β-lactam biosynthetic intermediates

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