Biosynthesis of cephalosporin antibiotics involves an expansion of the five-membered thiazolidine ring of penicillin N to the six-membered dihydrothiazine ring of deacetoxycephalosporin C by a deacetoxycephalosporin C synthetase (DAOCS) enzyme activity. Hydroxylation of deacetoxycephalosporin C to form deacetylcephalosporin C by a deacetylcephalosporin C synthetase (DACS) activity is the next step in biosynthesis of cephalosporins. In Cephalosporium acremonium, both of these catalytic activities are exhibited by a bifunctional enzyme, DAOCS-DACS, encoded by a single gene, cefEF. In Streptomyces clavuligerus, separable enzymes, DAOCS (expandase) and DACS (hydroxylase), catalyze these respective reactions. We have cloned, sequenced, and expressed in E. coli an S. clavuligerus gene, designated cefE, which encodes DAOCS but not DACS. The deduced amino acid sequence of DAOCS from S. clavuligerus (calculated Mr of 34,519) shows marked similarity (approximately 57%) to the deduced sequence of DAOCS-DACS from C. acremonium; however, the latter sequence is longer by 21 amino acid residues.
Isopenicihlin N isomerase (epimerase) has been purified from Streptomyces clavuligerus, and the amino acid sequence of the N-terminus has been determined. By using single oligonucleotide probes based on high GC codon bias ("guessmers"), the translation start codons were determined for two successive genes in the P-lactam-biosynthetic pathway and mapped within a 3.6-kilobase-pair KpnI restriction fragment. The epimerase gene (cel)) was located immediately upstream of the deacetoxycephalosporin C synthetase (expandase) gene (cefE) that was characterized previously. celD was sequenced and expressed in Escherichia coli; the resulting cell extracts contained epimerase activity. Western immunoblots demonstrated that a protein comigrated with purified S. clavuligerus epimerase at 44 kilodaltons. cel) and cejE were separated by an 81-base-pair segment. The DNA sequence upstream of the epimerase gene had a high AT content, suggestive of a promoter region. Primer extension analysis of S. clavuligerus mRNA showed that the start of transcription occurred approximately 130 base pairs upstream of the epimerase translation start site; Northern (RNA blot) analysis revealed a hybridization signal large enough to code for both epimerase and expandase, and nuclease Si protection assays showed that a single message may code for epimerase, expandase, and another ulnknown protein. When cef) and cejE were placed in an expression vector, concomitant synthesis of both epimerase and expandase occurred in E. coli.,-Lactam antibiotics are distinguished by the ,3-lactam ring plus either a five-membered thiazolidine ring (present in the penicillins) or the "expanded" six-membered dihydrothiazine structure (found in the cephalosporins). This is the main chemical difference between the core structures of these antibiotics. The pathway for these two classes of 13-lactams have common steps in their synthesis up to the production of isopenicillin N ( Fig. 1) (7, 11). The first step that commits the pathway to the production of cephalosporinlike antibiotics is the isomerization of isopenicillin N, the product of isopenicillin N synthetase (cyclase), to penicillin N (10, 12). This activity has also been referred to as epimerase and is not found in organisms (e.g., Penicillium chrysogenum) that produce hydrophobic penicillins (7).In a previous report (13) we characterized the gene (cefE) for deacetoxycephalosporin C synthetase (DAOCS; expandase) and expressed the gene in Escherichia coli. These E. coli extracts contained expandase activity. The gene was isolated from a cosmid library with a single hybridization probe based on the N-terminal amino acid sequence of the purified protein and the strict codon bias observed for streptomycetes (24). We have also shown by differential hybridization to these cosmids that genes for the cyclase and expandase enzymes are closely linked in S. clavuligerus (13,21). This potential for linkage of all the genes for the biosynthesis of cephalosporins led us to characterize the molecular organization of the epimerase ...
We have studied the secretion and processing of Staphylococcus aureus nuclease in Bacillus subtilis. We show that the initial species of nuclease found in the cell supernatants during short-term radioactive labeling (pulse-chase) had a molecular weight of approximately 18,800 and comigrated in a sodium dodecyl sulfate-polyacrylamide gel with staphylococcal nuclease B. This nuclease B form was processed to the mature nuclease A extracellularly by a phenylmethylsulfonyl fluoride-sensitive protease. The nuclease B-processing site is a consensus signal peptidase site, and the processing of nuclease B was coupled to secretion as judged by pulse-chase experiments. The nuclease A was shown by microsequencing of the N terminus to be 2 amino acid residues shorter than the nuclease A descriled for S. aureus Foggi. The nuclease B form was still the first species found in the culture supernatant after removal of the N-terminal 26 amino acids of the native 60-amino-acid signal peptide. However, removal of the N-terminal 72 amino acids abolishes secretion of any nuclease form and leads to the intracellular accumulation of nuclease.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.