Non-radioactive assay and HPLC analysis of cephalosporin C 7?-methoxylation by cell-free extracts of Streptomyces clavuligerus

Xiao, X. W.; Bowers, Raymond J.; Shin, Heesook; Wolfe, Saul; Demain, Arnold L.

doi:10.1007/bf00169897

Cited by 7 publications

(6 citation statements)

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“…Escherichia coli W 3110, which is resistant to cephalosporin C, has been reported to be sensitive to cephamycin C and 7K-methoxycephalosporin C [10]. Because E. coli W 3110 is sensitive to 7K-demethoxycefminox (MIC 0.78 Wg ml 31 ), it cannot be used for the bioassay of the reaction mixture.…”

Section: Resultsmentioning

confidence: 99%

Synthesis of cefminox by cell-free extracts of Streptomyces clavuligerus

Kim

2000

FEMS Microbiology Letters

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In vitro synthesis of cefminox by cell-free extracts of Streptomyces clavuligerus was investigated using K-ketoglutarate, L-ascorbic acid, FeSO 4 , S-adenosyl-L-methionine, and 7K-demethoxycefminox as the substrates. The formation of cefminox was detected both by a biological assay with Proteus vulgaris GN 76/C-1 and by high performance liquid chromatography. Although the conversion rate of 7K-demethoxycefminox to cefminox was observed to be quite low, it still demonstrated the potential for an enzymatic process to replace the chemical steps which are currently in use for the production of cefminox. ß

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Section: Resultsmentioning

confidence: 99%

Synthesis of cefminox by cell-free extracts of Streptomyces clavuligerus

Kim

2000

FEMS Microbiology Letters

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“…Excess SAM not retained in the QAE-Sephadex column eluted at 3.5 min. One of the labeled peaks (retention time, 17.7 min) was identified as 7-methoxycephalosporin C (19).…”

Section: Characterization Of Orf7 and Orf8mentioning

confidence: 99%

“…4. The reaction products were separated by HPLC using a Waters Bondapack C 18 column; the elution was made with a flow rate of 1.5 ml and a gradient of methanol as follows: 0 to 20 min, 0% methanol; 30 min, 5% methanol; 40 min, 10% methanol (19). Under these conditions, the retention times of the substrate and products were as follows: cephalosporin C, 34 min; 7-hydroxycephalosporin C, 14.2 min; 7-methoxycephalosporin C, 17.7 min.…”

Section: Characterization Of Orf7 and Orf8mentioning

confidence: 99%

A two-protein component 7 alpha-cephem-methoxylase encoded by two genes of the cephamycin C cluster converts cephalosporin C to 7-methoxycephalosporin C

et al. 1995

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Two genes, cmcI and cmcJ, corresponding to open reading frames 7 and 8 (ORF7 and ORF8) of the cephamycin C cluster of Nocardia lactamdurans encode enzymes that convert cephalosporin C to 7-methoxycephalosporin C. Proteins P7 and P8 (the products of ORF7 and ORF8 expressed in Streptomyces lividans) introduce the methoxyl group at C-7 of the cephem nucleus. Efficient hydroxylation at C-7 and transfer of the methyl group from S-adenosylmethionine require both proteins P7 and P8, although P7 alone shows weak C-7 hydroxylase activity and strong cephalosporin-dependent NADH oxidase activity. Both P7 and P8 appear to be synthesized in a coordinated form by translational coupling of cmcI and cmcJ. Protein P7 contains domains that correspond to conserved sequences in cholesterol 7␣-monooxygenases and to the active center of Omethyltransferases by comparison with the crystal structure of catechol-O-methyltransferase. Protein P8 may act as a coupling protein for efficient hydroxylation at C-7 in a form similar to that of the two-component system of Pseudomonas putida p-hydroxyphenylacetate-3-hydroxylase.Cephamycin C is synthesized in Nocardia lactamdurans and Streptomyces clavuligerus from precursor amino acids L-␣-aminoadipic acid, L-cysteine, L-valine, and L-methionine. The first three amino acids are linked to form the corresponding tripeptide by the multienzyme ␣-aminoadipyl-cysteinyl-valine synthetase (2, 5), which is encoded by the pcbAB gene (10). The ␣-aminoadipyl-cysteinyl-valine tripeptide is cyclized to form isopenicillin N, and this intermediate is epimerized to form penicillin N, which is later converted to deacetoxycephalosporin C by deacetoxycephalosporin C synthase (expandase). The genes encoding these three enzymatic steps, pcbC, cefD, and cefE, in N. lactamdurans are known to be clustered with lat (which encodes lysine-6-aminotransferase) and pcbAB (9, 11). Further reactions are involved in the synthesis of the C-7 methoxyl group and in the attachment of the carbamoyl group at C-3Ј. Little information is available about the so-called late genes, which convert deacetoxycephalosporin C into cephamycin C. Deacetoxycephalosporin C is known to be hydroxylated to deacetylcephalosporin C in S. clavuligerus by an ␣-ketoglutarate-requiring 3Ј-methylcephem hydroxylase (4, 16). Deacetylcephalosporin C is converted into O-carbamoyldeacetylcephalosporin C by an O-carbamoyltransferase that transfers a carbamoyl group from carbamoylphosphate to the 3Ј-hydroxyl group of deacetylcephalosporin C (6). The methoxyl group at C-7 in the cephamycins derives from molecular oxygen (15) and methionine (18) by the apparent action of an oxygenase and a methyltransferase.At least three types of oxygenases are involved in hydroxylations of microbial metabolites. The first class are ␣-ketoglutarate dependent and require Fe 2ϩ ions to introduce one of the oxygen atoms from O 2 into the substrate (1). A second class of flavin monooxygenases (e.g., p-hydroxybenzoate hydroxylase, salicylate hydroxylase, phenol hydroxylase, melilolate hydro...

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“…On the other hand, the cloning of the individual genes involved in 7a-methoxylation of cephalosporins has not been reported, although Chen et al (1) have described the cloning and expression of a cluster of genes for cephamycin C production from Streptomyces cattleya in the nonproducer Streptomyces lividans. Streptomyces clavuligerus cell-free enzymatic reactions indicate that methoxylation by actinomycetes occurs in two steps (8,12,18). The first step is the hydroxylation of cephalosporin at C-7, and the second step is methylation.…”

mentioning

confidence: 99%

“…The flasks were incubated on a rotary shaker (30°C, 250 rpm) for 3 days. The mycelium was collected by centrifugation (15 min, 2,200 x g) to prepare cell extracts for analysis of CH enzymatic activity as described previously (18,19). The activity was measured by a high-performance liquid chromatography (HPLC) assay which detected the formation of 7at-hydroxycephalosporin C (HCPC) from the substrate cephalosporin C (CPC) in a cell-free enzymatic reaction.…”

mentioning

confidence: 99%

Cloning of a Streptomyces clavuligerus DNA fragment encoding the cephalosporin 7 alpha-hydroxylase and its expression in Streptomyces lividans

Xiao

Hintermann

Häusler

et al. 1993

Antimicrob Agents Chemother

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A 26-mer DNA probe was designed from N-terminal sequence data for the cephalosporin 7a-hydroxylase (CH) of Streptomyces clavuligerus NRRL 3585 and used to screen a DNA library from this organism. The library was constructed in the AGEM-1l phage system. After plaque purification and reprobing, positive recombinant phages were chosen for further analysis. Characterization of the cloned DNA by restriction mapping and Southern hybridization showed that a 1.5-kb SailI fragment hybridized to the probe. Polymerase chain reaction assays using this fragment as a template and the probe as a primer indicated that the fragment carries the entire putative CH gene (cmcl). This was confirmed through the expression of CH enzymatic activity when the fragment was introduced into Streptomyces lividans. A putative 13-lactamase activity was detected in S. lividans.Some actinomycetes produce 7a-methoxylated cephalosporins, which are named cephamycins. Methoxylation of cephalosporins increases their inhibitory effect on transpeptidase(s) involved in bacterial cell wall synthesis (7) and reduces inactivation by 3-lactamases (2), making the cephamycins important clinical antibiotics. As a representative of cephamycins, cephamycin C exhibits activity against many cephalosporin-resistant bacteria (11). The cephalosporinproducing fungi and the cephamycin-producing actinomycetes share a biosynthetic pathway from L-a-aminoadipate, L-cysteine, and L-valine to deacetylcephalosporin C (3). However, the cephalosporin-producing fungi lack the methoxylation system to convert cephalosporins into cephamycins. Many, if not all, biosynthetic genes involved in the common pathway have been cloned from fungi and actinomycetes. The cloning of the genes for cyclase, epimerase, expandase, and deacetoxycephalosporin C hydroxylase has been reviewed elsewhere (14). More recently, the cloning of the oa-aminoadipyl-cysteinyl-valine (ACV) synthetase gene from Cephalosporium acremonium has also been reported (4, 10). On the other hand, the cloning of the individual genes involved in 7a-methoxylation of cephalosporins has not been reported, although Chen et al. (1) have described the cloning and expression of a cluster of genes for cephamycin C production from Streptomyces cattleya in the nonproducer Streptomyces lividans. Streptomyces clavuligerus cell-free enzymatic reactions indicate that methoxylation by actinomycetes occurs in two steps (8,12,18). The first step is the hydroxylation of cephalosporin at C-7, and the second step is methylation. Cephalosporin 7a-hydroxylase (CH), which catalyzes the first step, was purified to near homogeneity from S. clavuligerus NRRL 3585 (19). In the present study, using a degenerate mixed-oligonucleotide probe designed * Corresponding author.

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Non-radioactive assay and HPLC analysis of cephalosporin C 7?-methoxylation by cell-free extracts of Streptomyces clavuligerus

Cited by 7 publications

References 7 publications

Synthesis of cefminox by cell-free extracts of Streptomyces clavuligerus

Synthesis of cefminox by cell-free extracts of Streptomyces clavuligerus

A two-protein component 7 alpha-cephem-methoxylase encoded by two genes of the cephamycin C cluster converts cephalosporin C to 7-methoxycephalosporin C

Cloning of a Streptomyces clavuligerus DNA fragment encoding the cephalosporin 7 alpha-hydroxylase and its expression in Streptomyces lividans

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