Insights into the biosynthesis and stability of the lasso peptide capistruin.
Capistruin is a 19-residue ribosomally synthesized lasso peptide encoded by the capABCD gene cluster in Burkholderia thailandensis. It is composed of an N-terminal 9-residue macrolactam ring, through which the 10-residue C-terminal tail is threaded. Using a heterologous capistruin production system in Escherichia coli, we have generated 48 mutants of the precursor protein CapA to gain insights into capistruin biosynthesis. Only 4 residues (Gly1, Arg11, Val12, and Ile13) of the lasso sequence were found to be critical for maturation. Tandem mass spectrometric fragmentation studies of capistruin F16A/F18A proved Arg15 to be responsible for the trapping of the C-terminal tail. Substituting Arg15 and Phe16 by alanine revealed a temperature-sensitive capistruin derivative, which unfolds into a branched cyclic peptide upon heating. In conclusion, our global mutagenic approach revealed a low overall specificity of the biosynthetic machinery and important structure-stability correlations.
Knappe
Thomas A
TA
Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany.
Linne
Uwe
U
Robbel
Lars
L
Marahiel
Mohamed A
MA
D000595:Amino Acid Sequence; D019117:Burkholderia / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013696:Temperature
D010455:Peptides; D011994:Recombinant Proteins; C533129:capistruin