A spectrophotometric assay for deacetoxycephalosporin C synthase

Baldwin, Jack E.; Crabbe, M. James C.

doi:10.1016/0014-5793(87)80087-x

Cited by 18 publications

(10 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The structure and function of DAOCS have been partially elucidated by a series of site-directed mutagenesis experiments [6 -8] and complementary X-ray crystal structures [3,9]. A number of assays for DAOCS and other oxygenases have been used in the past, including monitoring production of succinate [10,11] or carbon dioxide [6] from 2-oxoglutarate, and bioactivity [12], high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) assays [3,13] for production of cephems. The inherent drawback of these methods is that they are discontinuous and only yield one point on the time course curve describing the enzyme kinetic reaction.…”

Section: Introductionmentioning

confidence: 99%

“…There also appears to be a discrepancy between the in vitro results and the in vivo production of cephems; e. g. penicillin G (2a) is a poor substrate for recombinant DAOCS in vitro [3], but was not expanded at all to the phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA) (2b) in recombinant Penicillium chrysogenum [2]. A spectrophotometric assay for DAOCS has previously been described [10] but its potential uses were never developed. This paper reports a novel direct spectrophotometric assay for DAOCS based on continuous monitoring of absorbance at 260 nm due to the production of the deacetoxycephem ring.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase

Dubus¹,

Lloyd²,

Lee³

et al. 2001

CMLS, Cell. Mol. Life Sci.

View full text Add to dashboard Cite

Deacetoxycephalosporin C synthase from Streptomyces clavuligerus catalyses the conversion of the five-membered penicillin ring to the unsaturated six-membered cephem ring of deacetoxycephalosporin C. The effects on enzyme activity of the penicillin substrate sidechain and various cofactors were investigated using a continuous spectrophotometric assay. The conversion of penicillin G to phenylacetyl-7-aminodeacetoxycephalo sporanic acid (G-7-ADCA) was confirmed, and further details of the reaction were elucidated. The conversion of ampicillin to cephalexin was faster than that of acetyl-6-APA to acetyl-7-ADCA kcat = 0.120 +/- 0.001 s(-1) versus 0.035 +/- 0.001 s(-1), but they had similar Km values: 4.86 +/- 0.12 and 3.28 +/- 0.26 mM, respectively. Amoxycillin and penicillin V were also converted at low levels. Conversion was not detected for penicillanate, 6-aminopenicillanate, carbenicillin, temocillin, ticarcillin or benzylpenicilloic acid, suggesting that the enzyme has a relatively strict selectivity for the sidechain of the penicillin substrate.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase

Dubus¹,

Lloyd²,

Lee³

et al. 2001

CMLS, Cell. Mol. Life Sci.

View full text Add to dashboard Cite

show abstract

“…In addition, these assays are discontinuous and, hence, a large number of samples are required for kinetic studies. A continuous direct spectrophotometric assay for determination of penicillin N conversion by DAOCS was Wrst reported by Baldwin and Crabbe [8]. However, this assay contained sodium azide as one of the components in the reaction mixture, which probably limited its application.…”

Section: Biochemical Characterization Of Daocs: Enzymatic Activity Anmentioning

confidence: 99%

Directed evolution and rational approaches to improving Streptomyces clavuligerus deacetoxycephalosporin C synthase for cephalosporin production

Goo

Chua

Sim

2009

J Ind Microbiol Biotechnol

View full text Add to dashboard Cite

It is approximately 60 years since the discovery of cephalosporin C in Cephalosporium acremonium. Streptomycetes have since been found to produce the structurally related cephamycin C. Studies on the biosynthetic pathways of these two compounds revealed a common pathway including a step governed by deacetoxycephalosporin C synthase which catalyses the ring-expansion of penicillin N to deacetoxycephalosporin C. Because of the therapeutic importance of cephalosporins, this enzyme has been extensively studied for its ability to produce these antibiotics. Although, on the basis of earlier studies, its substrate specificity was believed to be extremely narrow, relentless efforts in optimizing the in-vitro enzyme assay conditions showed that it is able to convert a wide range of penicillin substrates differing in their side chains. It is a member of 2-oxoglutarate-dependent dioxygenase protein family, which requires the iron(II) ion as a co-factor and 2-oxoglutarate and molecular oxygen as co-substrates. It has highly conserved HXDX( n ) H and RXS motifs to bind the co-factor and co-substrate, respectively. With advances in technology, the genes encoding this enzyme from various sources have been cloned and heterologously expressed for comparative analyses and mutagenesis studies. A high level of recombinant protein expression has also enabled crystallization of this enzyme for structure determination. This review will summarize some of the earlier biochemical characterization and describe the mechanistic action of this enzyme revealed by recent structural studies. This review will also discuss some of the approaches used to identify the amino acid residues involved in binding the penicillin substrate and to modify its substrate preference for possible industrial application.

show abstract

“…A spectrophotometric method similar to that reported previously by Baldwin and Crabbe (1987) was used. The reactions were performed at the same conditions as for bioassays.…”

Section: Spectrophotometric Assay Of Penicillin Conversionmentioning

confidence: 99%

“…Thus a more sensitive spectrophotometric method (Baldwin and Crabbe 1987) was used to verify the bioassay results. The spectrophotometric data are listed in Table 2.…”

Section: Mutation and Expression Of Wt And Mutant Acdaoc/dacsmentioning

confidence: 99%

Saturation mutagenesis of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase R308 site confirms its role in controlling substrate specificity

Tian

et al. 2010

Biotechnol Lett

View full text Add to dashboard Cite

Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C and the hydroxylation of the latter to deacetylcephalosporin C. The R308 residue located in close proximity to the C-terminus of acDAOC/DACS was mutated to the other 19 amino acids. In the resulting mutant pool, R308L, R308I, R308T and R308V showed significant improvement in their ability to convert penicillin analogs, thus confirming the role of R308 in controlling substrate selectivity, the four amino acids all possess short aliphatic sidechains that may improve hydrophobic interactions with the substrates. The mutant R308I showed the highest reactivity for penicillin G, with 3-fold increase in k(cat)/K(m) ratio and 7-fold increase in relative activity.

show abstract

A spectrophotometric assay for deacetoxycephalosporin C synthase

Cited by 18 publications

References 13 publications

Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase

Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase

Directed evolution and rational approaches to improving Streptomyces clavuligerus deacetoxycephalosporin C synthase for cephalosporin production

Saturation mutagenesis of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase R308 site confirms its role in controlling substrate specificity

Contact Info

Product

Resources

About