Dithiothreitol reactivates desacetoxycephalosporin C synthetase after inactivation

Lübbe, Claus; Wolfe, Saul; Demain, Arnold L.

doi:10.1016/0141-0229(85)90115-2

Cited by 12 publications

(5 citation statements)

References 11 publications

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“…DTT and ascorbate contribute to the stability and optimal catalysis (Table 2) of the expandase, presumably by keeping iron in the reduced form or by protecting essential sulfhiydryl groups of the enzyme (16,27). Greater expression of both activities with Fe2+ than with Fe3+ in the presence of DTT or ascorbate suggests that, in optimal catalysis by the expandase/hydroxylase, one common function of DTT and ascorbate is iron reduction.…”

Section: Resultsmentioning

confidence: 99%

“…Except for their respective P-lactam substrates, both enzymes required a-KG, Fe2+, and 02 and were stimulated by ascorbate, DTT, and ATP. The common requirements and stimulations were shown for partially purified (14,16,27) as well as highly purified ( (20,25) in the expandasecatalyzed reaction. The tight stoichiometric conversion (1:1) of penicillin N to DAOC plus DAC by the expandase/ hydroxylase (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Dotzlaf¹,

Yeh²

1987

J Bacteriol

121

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“…For example, DTT added at 5 mM stimulated the expandase activity detected in the cellfree extracts of S. clavuligerus (Lübbe et al 1985) whereas Cho et al (1998) used 16 mM DTT in reaction mixture concentration of the substrate, co-substrate (a-ketoglutarate) and FeSO 4 , and also by using a high concentration of cells, conversion of penicillin G to DAOG was markedly improved. Using such conditions, they showed activity on penicillin G and 14 other penicillins including ampicillin with cell-free extract of Streptomyces clavuligerus NP1.…”

Section: Fractionation Of Cell-free Extracts and Sds-pagementioning

confidence: 94%

“…Despite the obvious importance of the ring expansion reaction, earlier studies on DAOCS have often been impeded by poor enzyme stability and the difficulty in obtaining a large amounts of purified enzyme (Lübbe et al 1985). Enzymes involved in the biosyntheses of secondary metabolites usually exhibit broad substrate specificities.…”

Section: Introductionmentioning

confidence: 99%

Expandase-like activity mediated cell-free conversion of ampicillin to cephalexin by Streptomyces sp. DRS I

2009

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Cell-free extracts of Streptomyces sp. DRS I converted ampicillin to cephalexin, presumably due to the activity of the enzyme, expandase. The extract was fractionated and characterized by colorimetric and chromatographic measurements coupled with disc-agar diffusion bioassay against an ampicillin-resistant, cephalexin-sensitive E. coli strain. Though expandase could not be identified, the presence of a hitherto unreported expandase in Streptomyces sp. DRS I is suggested.

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Synthesis of β‐Lactams (Cephalosporins) by Bioconversion

Barredo¹,

Rodríguez‐Sáiz²,

Adrio

et al. 2010

Amino Acids, Peptides and Proteins in Organic Chemistry

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Ring expansion of the five-membered thiazolidine ring of the intermediate penicillin N to the six-membered dihydrothiazine ring of deacetoxycephalosporin C (DAOC) (Figure 4.1) was discovered by Kohsaka and Demain [1] working with cell-free extracts of Acremonium chrysogenum (formerly Cephalosporium acremonium). DAOC synthase (deacetoxycephalosporin C synthaseDAOCS; also known as expandase) is a crucial enzyme responsible for converting penicillins to cephalosporins. In cephalosporin producers, A. chrysogenum and Streptomyces clavuligerus, expandase is an iron-and a-ketoglutarate-dependent enzyme. In the bacterium, S. clavuligerus, the subsequent hydroxylation of the methyl group of DAOC to give deacetylcephalosporin C (DAC) is catalyzed by a closely related enzyme, DAC synthase (deacetylcephalosporin C synthase DACS), whereas in the fungus A. chrysogenum, the activities of expandase and hydroxylase reside in a single functional protein (Figure 4.1).The bifunctional expandase of A. chrysogenum is a monomer with a molecular weight of 41 kDa and an isoelectric point (pI) of 6.3 [2]. It is a bifunctional enzyme that catalyzes not only ring expansion but also the hydroxylation of the methyl group of DAOC to DAC. Expandase exhibited properties of a-ketoglutarate-linked dioxygenases [3]. Ring expansion activity was markedly increased by ascorbic acid, Fe þ 2 [4, 5], a-ketoglutarate [4, 6], oxygen [6,7], dithiothreitol (DTT), and ATP. The enzyme showed an absolute requirement for a-ketoglutarate [4, 8-10] and this cosubstrate could not be replaced by chemically related compounds such as a-ketoadipate, pyruvate, oxaloacetate, glutamate, succinate, a-ketovalerate, or a-ketobutyrate [11][12][13]. The properties of the expandase are similar to those of other a-ketoglutarate-dependent dioxygenases that incorporate one atom of oxygen into a-ketoglutarate to form succinate and the other atom into the product molecule. However, expandase differs in that no oxygen atom appears in its ring expansion product, DAOC. When isopenicillin N, penicillin G, penicillin V, ampicillin, and 6-aminopenicillanic acid (6-APA) were tested as substrate analogs of penicillin N, no ring expansion was observed [2,11,14].

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Dithiothreitol reactivates desacetoxycephalosporin C synthetase after inactivation

Cited by 12 publications

References 11 publications

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

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

Expandase-like activity mediated cell-free conversion of ampicillin to cephalexin by Streptomyces sp. DRS I

Synthesis of β‐Lactams (Cephalosporins) by Bioconversion

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