Purification and characterization of cephalosporin 7α-hydroxylase from <i>Streptomyces clavuligerus</i>

Xiao, X. W.; Wolfe, Saul; Demain, Arnold L.

doi:10.1042/bj2800471

Cited by 25 publications

(17 citation statements)

References 10 publications

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“…In Cephalosporium acremonium a single, bifunctional protein, deacetoxy/deacetylcephalosporin C synthase (DAOC/DAC synthase), performs both reactions (2)(3)(4). S. clavuligerus also contains a 7␣-hydroxylase, which is involved in the biosynthesis of cephamycin C from DAC (5).…”

Section: Deacetoxycephalosporin C Synthase (Daocs)mentioning

confidence: 99%

Alteration of the Co-substrate Selectivity of Deacetoxycephalosporin C Synthase

Lee

Lloyd²,

Clifton³

et al. 2001

Journal of Biological Chemistry

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Deacetoxycephalosporin C synthase is an iron(II) 2-oxoglutaratedependent oxygenase that catalyzes the oxidative ring-expansion of penicillin N to deacetoxycephalosporin C. The wild-type enzyme is only able to efficiently utilize 2-oxoglutarate and 2-oxoadipate as a 2-oxoacid co-substrate. Mutation of arginine 258, the side chain of which forms an electrostatic interaction with the 5-carboxylate of the 2-oxoglutarate co-substrate, to a glutamine residue reduced activity to about 5% of the wild-type enzyme with 2-oxoglutarate. However, other aliphatic 2-oxoacids, which were not cosubstrates for the wild-type enzyme, were utilized by the R258Q mutant. These 2-oxoacids "rescued" catalytic activity to the level observed for the wild-type enzyme as judged by penicillin N and G conversion. These cosubstrates underwent oxidative decarboxylation as observed for 2-oxoglutarate in the normal reaction with the wild-type enzyme. Crystal structures of the iron(II)-2-oxo-3-methylbutanoate (1.5 Å), and iron(II)-2-oxo-4-methylpentanoate (1.6 Å) enzyme complexes were obtained, which reveal the molecular basis for this "chemical co-substrate rescue" and help to rationalize the co-substrate selectivity of 2-oxoglutaratedependent oxygenases. Deacetoxycephalosporin C synthase (DAOCS)1 is an iron(II) and 2-oxoglutarate-dependent oxygenase that catalyzes the conversion of penicillin N to deacetoxycephalosporin C in the biosynthesis of cephem antibiotics in Streptomyces clavuligerus (1). The subsequent hydroxylation of DAOC to deacetylcephalosporin C (DAC) is catalyzed by a closely related enzyme, deacetylcephalosporin C synthase (DACS). In Cephalosporium acremonium a single, bifunctional protein, deacetoxy/deacetylcephalosporin C synthase (DAOC/DAC synthase), performs both reactions (2-4). S. clavuligerus also contains a 7␣-hydroxylase, which is involved in the biosynthesis of cephamycin C from DAC (5).DAOCS is a member of the iron(II) and 2-oxoglutarate-dependent oxygenase family, which catalyze a wide variety of oxidative reactions (6, 7). DAOCS, DACS, and DAOC/DAC synthase belong to a subgroup of more closely related enzymes, which have significant primary sequence homology to one another (6). Also included in this group are two enzymes which do not use 2-oxoglutarate as a co-substrate, isopenicillin N synthase (IPNS), the iron-dependent oxidase responsible for formation of the penicillin nucleus, and 1-amino-1-carboxycyclopropane oxidase (ACCO) which catalyzes the last step during ethylene biosynthesis in plants.Mechanistic understanding of iron(II), 2-oxoglutaratedependent oxygenases have been significantly advanced by the recently determined crystal structures of DAOCS (1,8,9) and clavaminic acid synthase (10). The DAOCS crystal structures revealed the presence of a number of arginine residues within the active site, with arginine 258 (part of a conserved RXS motif) being involved in co-substrate binding (1,8). The equivalent RXS motif residues in Aspergillus nidulans IPNS, arginine 281, and serine 283, bind the ␣-carboxylate gro...

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Section: Deacetoxycephalosporin C Synthase (Daocs)mentioning

confidence: 99%

Alteration of the Co-substrate Selectivity of Deacetoxycephalosporin C Synthase

Lee

Lloyd²,

Clifton³

et al. 2001

Journal of Biological Chemistry

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“…Initial evidence showed that P7 and P8 copurify after Blue Sepharose (Pharmacia) purification. Xiao et al (21) reported the purification of a major protein band (32 kDa) that correlated with 7␣-hydroxylase activity. A second band with a slightly lower molecular mass was seen in their gels after Blue Sepharose purification.…”

Section: Characterization Of Orf7 and Orf8mentioning

confidence: 99%

“…In summary, P7 is required for hydroxylase activity and may be equivalent to the 32-kDa protein isolated by Xiao et al (21). Importantly, we have found another coding sequence (ORF8) that, when present with ORF7 in S. lividans, yields methoxylated cephalosporin C. Molecular genetic evidence and preliminary biochemical evidence indicate that P7 and P8 are necessary for efficient methoxylation of cephalosporin; the methoxylation system is also reminiscent of a two-component hydroxylase, such as the p-hydroxyphenylacetate-3-hydroxylase from P. putida that includes a flavoprotein with substrate-stimulated NADH oxidase (P7) and a coupling protein (P8) that is crucial for coupling and hydroxylation of the substrate.…”

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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“…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.…”

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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Purification and characterization of cephalosporin 7α-hydroxylase from Streptomyces clavuligerus

Cited by 25 publications

References 10 publications

Alteration of the Co-substrate Selectivity of Deacetoxycephalosporin C Synthase

Alteration of the Co-substrate Selectivity of Deacetoxycephalosporin C Synthase

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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