The pipecolate-incorporating enzyme for the biosynthesis of the immunosuppressant rapamycin: Nucleotide sequence analysis, disruption and heterologous expression of rapP from Streptomyces hygroscopicus.
König, Ariane; Schwecke, T.; Molnár, I.et al.
1997 • In European Journal of Biochemistry, 247 (2), p. 526-534
[en] An open reading frame (rapP) encoding the putative pipecolate-incorporating enzyme (PIE) has been identified in the gene cluster for the biosynthesis of rapamycin in Streptomyces hygroscopicus. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetases. Disruption of rapP by phage insertion abolished rapamycin production in S. hygroscopicus, and the production of the antibiotic was specifically restored upon loss of the inserted phage by a second recombination event. rapP was expressed in both Escherichia coli and Streptomyces coelicolor, and recombinant PIE was purified to homogeneity from both hosts. Although low-level incorporation of [14C]beta-alanine into recombinant PIE isolated from E. coli was detected, formation of the covalent acylenzyme intermediate could only be shown with the PIE from S. coelicolor, suggesting that while the recombinant PIE from S. coelicolor was phosphopantetheinylated, only a minor proportion of the recombinant enzyme from E. coli was post-translationally modified.
Disciplines :
Biochemistry, biophysics & molecular biology
Identifiers :
UNILU:UL-ARTICLE-2011-403
Author, co-author :
König, Ariane ; University of Cambridge, UK > Cambridge Centre for Molecular Recognition > Department of Biochemistry
Schwecke, T.; University of Cambridge, UK > Cambridge Centre for Molecular Recognition > Department of Biochemistry
Molnár, I.; University of Cambridge > Cambridge Centre for Molecular Recognition > Department of Biochemistry
Böhm, G. A.; University of Cambridge > Cambridge Centre for Molecular Recognition > University Chemical Laboratory
Lowden, P. A. S.; University of Cambridge > Cambridge Centre for Molecular Recognition > University Chemical Laboratory
Staunton, J.; University of Cambridge > Cambridge Centre for Molecular Recognition > University Chemical Laboratory
Leadlay, P. F.; University of Cambridge > Cambridge Centre for Molecular Recognition > Department of Biochemistry
External co-authors :
yes
Language :
English
Title :
The pipecolate-incorporating enzyme for the biosynthesis of the immunosuppressant rapamycin: Nucleotide sequence analysis, disruption and heterologous expression of rapP from Streptomyces hygroscopicus.