Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase

Dubus, Alain; Lloyd, Matthew D.; Lee, H.-J.; Schofield, Christopher J.; Baldwin, Jack E.; Frère, Jean Marie

doi:10.1007/pl00000904

Cited by 28 publications

(29 citation statements)

References 34 publications

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“…The results also suggested that 6-APA which is devoid of an aromatic side chain might lead to packing de¢ciency in the catalytic pocket of DAOCSs, and thus hamper the catalytic e⁄ciency of the DAOCSs. Since, Dubus et al have suggested that the acetyl group might be the minimal side chain amenable for the ring expansion of DAOCS [18], the outcomes observed were expected. In contrast, the bulky side chain of oxacillin might increase the steric hindrance in the catalytic center that could disrupt the enzymatic action of DAOCSs.…”

Section: Discussionmentioning

confidence: 96%

“…The conversions of these penicillin N analogues by the recombinant cDAOC/ DACS have not yet been reported thus far. Dubus et al [18] a⁄rmed that scDAOCS was unable to convert carbenicillin due to the relatively strict selectivity for the side chain of the penicillin substrate. However, in this study, the conversion of carbenicillin is clearly catalyzed by recombinant scDAOCS, sjDAOCS and cDAOC/DACS.…”

Section: Discussionmentioning

confidence: 99%

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Deacetoxycephalosporin C synthase isozymes exhibit diverse catalytic activity and substrate specificity

Chin

2003

FEMS Microbiology Letters

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The biosynthesis of cephalosporins involving a thiozolidine ring expansion is catalyzed by deacetoxycephalosporin C synthase (DAOCS). In this study, three DAOCS isozymes were cloned and expressed as active enzymes together with Streptomyces jumonjinensis DAOCS that was newly isolated and partially characterized. The enzymes showed excellent substrate conversion for penicillin G, phenethicillin, ampicillin and carbenicillin, but they were less effective in the ring expansion of penicillin V, amoxicillin and metampicillin. Streptomyces clavuligerus DAOCS was the most active among the recombinant enzymes. The results also showed that truncation of 20 amino acids at the C-terminus of the Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase polypeptide did not affect penicillin ring expansion.

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Deacetoxycephalosporin C synthase isozymes exhibit diverse catalytic activity and substrate specificity

Chin

2003

FEMS Microbiology Letters

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“…Therefore, AlkB inhibition at high 2OG concentrations is unlikely to be a result of Fe II chelation by 2OG. The 2OG-dependent dioxygenases deacetoxycephalosporin C synthase and thymine-7-hydroxylase have also been observed to be inhibited by high concentrations of 2OG (33,38). Enzyme inhibition by high substrate concentrations is often thought to be a result of two molecules of substrate binding to the enzyme to produce an inactive complex (39).…”

Section: Resultsmentioning

confidence: 99%

The Selectivity and Inhibition of AlkB

Welford¹,

Schlemminger²,

McNeill³

et al. 2003

Journal of Biological Chemistry

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AlkB is one of four proteins involved in the adaptive response to DNA alkylation damage in Escherichia coli and is highly conserved from bacteria to humans. Recent analyses have verified the prediction that AlkB is a member of the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family of enzymes. AlkB mediates repair of methylated DNA by direct demethylation of 1-methyladenine and 3-methylcytosine lesions. Other members of the Fe(II) and 2OG-dependent oxygenase family, including those involved in the hypoxic response, are targets for therapeutic intervention. Assays measuring 2OG turnover were used to investigate the selectivity of AlkB. 1-Methyladenosine, 1-methyl-2 -deoxyadenosine, 3-methylcytidine, and 3-methyl-2 -deoxycytidine all stimulated 2OG turnover by AlkB but were not demethylated indicating an uncoupling of 2OG and prime substrate oxidation and that oligomeric DNA is required for hydroxylation and subsequent demethylation. In contrast the equivalent unmethylated nucleosides did not stimulate 2OG turnover indicating that the presence of a methyl group in the substrate is important in initiating oxidation of 2OG. Stimulation of 2OG turnover by 1-methyladenosine was highly dependent on the presence of a reducing agent, ascorbate or dithiothreitol. Following the observation that AlkB is inhibited by high concentrations of 2OG, analogues of 2OG, including 2-mercaptoglutarate, were found to specifically inhibit AlkB. The flavonoid quercetin inhibits both AlkB and the 2OG oxygenase factor-inhibiting hypoxia-inducible factor (FIH) in vitro. FIH inhibition by quercetin occurs in the presence of excess iron indicating a specific interaction, while the inhibition of AlkB by quercetin is, predominantly, due to nonspecific iron chelation.The integrity of the genome is maintained by a set of DNA repair enzymes, including those that repair alkylation damage (1). DNA can be alkylated by a variety of agents that occur both exogenously and endogenously (2, 3). Alkylating agents are used in some chemotherapy treatments, and alkylated DNA bases have been detected in the urine of smokers (4). AlkB catalyzes demethylation of DNA and along with AlkA, AidB, and Ada is one of four proteins involved in the adaptive response to alkylation damaged DNA in Escherichia coli (5). Ada, which has been termed the "suicidal repair protein," contains two active sites, one of which demethylates O 6 -methylguanine by irreversible transfer of the methyl group to a cysteine (5, 6).The second active site removes methyl groups from S-methyl phosphoesters by nucleophilic methylation of another cysteine residue (5,7,8). Methylation of the latter cysteine converts Ada into a strong transcriptional activator of both its own production and the other three proteins of the adaptive response (9). AlkA is a DNA glycosylase with a wide substrate selectivity, including excision of cytotoxic 3-methyladenine residues (10). AlkA "flips" the damaged base out of the DNA double helix by inserting Leu-125 into the position occupied by the damaged base (11)...

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“…Conversion of [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]-2-oxoglutarate was monitored using the reported assay (12) under the same cofactor conditions. Kinetic assays were performed using the reported spectrophotometric assay (27), assuming ⑀ ϭ 6100 M Ϫ1 cm Ϫ1 for the conversion of penicillin G to G-7-ADCA. Large-scale incubations of penicillin and cephem substrates for product isolation were performed as reported previously (7,28) with appropriate modifications.…”

Section: Molecular Modeling Of Daoc/dacs and Dacs And Selection Of Mumentioning

confidence: 99%

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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Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase

Cited by 28 publications

References 34 publications

Deacetoxycephalosporin C synthase isozymes exhibit diverse catalytic activity and substrate specificity

Deacetoxycephalosporin C synthase isozymes exhibit diverse catalytic activity and substrate specificity

The Selectivity and Inhibition of AlkB

Controlling the Substrate Selectivity of Deacetoxycephalosporin/deacetylcephalosporin C Synthase

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