Enzymatic ring expansion of penicillins to cephalosporins: side chain specificity

Baldwin, Jack E.; Adlington, Robert M.; Coates, J. B.; Crabbe, M. James C.; Keeping, John W.; Knight, Graham; Nomoto, Takashi; Schofield, Christopher J.; Ting, Hong-Hoi

doi:10.1039/c39870000374

Cited by 15 publications

(5 citation statements)

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“…As expected (29,30), the results show that the penicillin substrate selectivity of recombinant DAOC/ DACS appears to be similar to that observed for DAOCS (7,27,28) with the exception that 6-␣-methylpenicillin is a substrate for DAOC/DACS but cannot be ring-expanded by DAOCS (31). Highest levels of ring-expansion activity for recombinant DAOC/DACS were observed with 6-␣-methylpenicillin N (31) (a close analog of the natural substrate, penicillin N) and penicillin G (29,30) with lower levels of activity observed with adipoyl-6-aminopenicillanic acid (29,30), ampicillin, and acetyl-6-aminopenicillanic acid (Table III). NMR analyses of incubations with cephem substrates showed conversion of DAOC (the ring-expansion product from penicillin N, the natural penam substrate) and G-7-ADCA (the ring-expanded product of penicillin G) to their corresponding hydroxylated products.…”

Section: Fig 1 Oxidative Reactions Carried Out By Daoc/dacs (C Acrsupporting

confidence: 54%

Controlling the Substrate Selectivity of Deacetoxycephalosporin/deacetylcephalosporin C Synthase

Lloyd

Lipscomb²,

Hewitson³

et al. 2004

Journal of Biological Chemistry

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Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutaratedependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with sitedirected mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (⌬310 mutant) enhanced ring-expansion of penicillin G by ϳ2-fold. A double mutant, ⌬310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The ⌬310/N305L/ M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K m ‫؍‬ 2.00 ؎ 0.47 compared with 6.02 ؎ 0.97 mM for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305.

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Section: Fig 1 Oxidative Reactions Carried Out By Daoc/dacs (C Acrsupporting

confidence: 54%

Controlling the Substrate Selectivity of Deacetoxycephalosporin/deacetylcephalosporin C Synthase

Lloyd

Lipscomb²,

Hewitson³

et al. 2004

Journal of Biological Chemistry

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“…21IB). 252,[306][307][308] A 2b-methyl-penam derivative (equivalent to penicillin N without the 2a-methyl group) was converted by DAOCS/DACS into a C-3-demethylated DAOC (Fig. 21IC).…”

Section: Biosynthesis Of the Hydrophobic Penicillinsisopenicillin Ami...mentioning

confidence: 99%

The enzymes of β-lactam biosynthesis

et al. 2013

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The β-lactam antibiotics and related β-lactamase inhibitors are amongst the most important small molecules in clinical use. Most, but not all, β-lactams including penicillins, cephalosporins, and clavulanic acid are produced via fermentation or via modification of fermented intermediates, with important exceptions being the carbapenems and aztreonam. The desire for more efficient routes to existing antibiotics and for access to new and synthetically challenging ones stimulates continued interest in β-lactam biosynthesis. We review knowledge of the pathways leading to β-lactam antibiotics focusing on the mechanisms, structures and biocatalytic applications of the enzymes involved.

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“…As DTT and ascorbate may both be serving the same function, to keep the iron in a reduced form and/or to maintain protein thiols in their reduced state [3], this may not be a serious problem, and the DTT concentration in the direct assay has been increased to l0 mM (from 1 mM in the bioassay). The direct spectrophotometric method has been used successfully in determining the substrate specificity of DAOC synthase with a number of structural variants of Pen N [11].…”

Section: Resultsmentioning

confidence: 99%

A spectrophotometric assay for deacetoxycephalosporin C synthase

Baldwin

Crabbe

1987

FEBS Letters

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A continuous direct spectrophotometric assay for deacetoxycephalosporin C synthase was developed, based on the absorption at 260 nm characteristic of the dihydrothiazine moiety of cephalosporins. Km values of 0.18 mM for penicillin N and 0.16 mM for ct-ketoglutarate were determined. A coupled assay using succinate thiokinase, pyruvate kinase and lactate dehydrogenase showed that succinate was a product of both deacetoxycephalosporin C synthase and hydroxylase reactions. The expandase reaction exhibited a 1:1.06 stoichiometry for deacetoxycephalosporin C and succinate.

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Enzymatic ring expansion of penicillins to cephalosporins: side chain specificity

Cited by 15 publications

References 0 publications

Controlling the Substrate Selectivity of Deacetoxycephalosporin/deacetylcephalosporin C Synthase

Controlling the Substrate Selectivity of Deacetoxycephalosporin/deacetylcephalosporin C Synthase

The enzymes of β-lactam biosynthesis

A spectrophotometric assay for deacetoxycephalosporin C synthase

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