Abstract
Pentalenolactone (PL), an antibiotic produced by Streptomyces arenae, is a potent inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The producer strain contains different isoforms of GAPDH: a PL-sensitive enzyme on nonproduction media and a PL-insensitive enzyme on production media. After induction of PL synthesis, the sensitive GAPDH disappears parallel to the disappearance of its activity, as shown by Western (immunoblot) hybridization. The two isoenzymes exhibit little immunological cross-reactivity and differ in size, amino acid composition, and several amino acid residues of their amino termini. Two different types of plasmids from a S. arenae genomic library, named pBRPLR1 and pBRPLR2, were cloned in Escherichia coli by selection for enhanced PL resistance. Both contain a GAPDH structural gene. Plasmid pBRPLR1 increases E. coli PL tolerance 7-fold, and plasmid pBRPLR2 increases it 30-fold. GAPDH from pBRPLR1 transformants shows biphasic PL inactivation kinetics. These cells contain PL-sensitive GAPDH from both E. coli and S. arenae. GAPDH from pBRPLR2 transformants tolerates higher PL concentrations than either E. coli or S. arenae PL-sensitive GAPDH but is less resistant than S. arenae PL-insensitive GAPDH. Nondenaturing polyacrylamide electrophoresis showed this GAPDH to be a hybrid of E. coli and S. arenae PL-insensitive GAPDH. The hybrid enzyme could be purified to homogeneity. Induction of the lacZ promoter of pUC subclones of both GAPDH genes had only a small effect on raising the level of intracellular GAPDH.
Highlights
Pentalenolactone (PL), an antibiotic produced by Streptomyces arenae, is a potent inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
The question of whether other mechanisms of resistance to this antibiotic exist in the producer strain remained open. We show that these enzymes are encoded by two genes with different regulation and that resistant Escherichia coli transformants express the PL-insensitive isoenzyme of S. arenae
Structural relationship of GAPDH isoenzymes. Both PLsensitive and PL-insensitive GAPDH isoenzymes were purified from S. arenae as described in Materials and Methods, and their amino acid compositions were compared (Table 1)
Summary
Pentalenolactone (PL), an antibiotic produced by Streptomyces arenae, is a potent inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Two different types of plasmids from a S. arenae genomic library, named pBRPLR1 and pBRPLR2, were cloned in Escherichia coli by selection for enhanced PL resistance GAPDH from pBRPLRl transformants shows biphasic PL inactivation kinetics These cells contain PL-sensitive GAPDH from both E. coli and S. arenae. The sesquiterpene antibiotic pentalenolactone (PL) selectively and irreversibly inactivates the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [Dglyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) of all procaryotic and eucaryotic organisms tested so far, binding covalently to the enzyme in a stoichiometric reaction [8, 9, 16, 25] It is a potent inhibitor of growth when glucose is the obligate carbon source. We show that these enzymes are encoded by two genes with different regulation and that resistant Escherichia coli transformants express the PL-insensitive isoenzyme of S. arenae
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
