B Walzel1, B Riederer, U Keller. 1. Max-Volmer-Institut für Biophysikalische Chemie und Biochemie Fachgebiet Biochemie und Molekulare Biologie Technische Universität Berlin Franklinstrasse 29, D-10587, Berlin-Charlottenburg, Germany.
Abstract
BACKGROUND: Previous analyses of the biosynthesis of the alkaloid cyclopeptides from the ergot fungus Claviceps purpurea were hampered by a lack of suitable systems for study in vitro, and this led to conflicting results concerning the mechanism of alkaloid cyclopeptide formation. Recently, D-lysergyl peptide synthetase (LPS) of the ergot fungus Claviceps purpurea, which assembles the non-cyclol precursors of the ergopeptines, has been partially purified and shown to consist of two polypeptide chains of 370 kDa (LPS 1) and 140 kDa (LPS 2); these contain all the sites necessary for the assembly of the D-lysergyl peptide backbone. The mechanism of D-lysergyl peptide synthesis remained unclear, however. RESULTS: We have identified the obligatory peptidic intermediates in D-lysergyl peptide synthesis and the sequential order of their formation. The two LPS subunits catalyze the formation of D-lysergyl mono-, di-, and tripeptides as enzyme-thioester intermediates, the formation of which appears to be irreversible. Peptide synthesis starts when D-lysergic acid binds to the LPS 2 subunit, which most probably occurs after the previous round of synthesis has been completed by the release of the end product from the LPS enzyme. CONCLUSIONS: We have shown that the mechanism of D-lysergyl peptide synthesis is an ordered process of successive acyl transfers on a multienzyme complex. This knowledge opens the way for enzymatic and genetic investigations into the formation of novel alkaloid cyclopeptides.
BACKGROUND: Previous analyses of the biosynthesis of the alkaloidcyclopeptides from the ergot fungus Claviceps purpurea were hampered by a lack of suitable systems for study in vitro, and this led to conflicting results concerning the mechanism of alkaloid cyclopeptide formation. Recently, D-lysergyl peptide synthetase (LPS) of the ergot fungus Claviceps purpurea, which assembles the non-cyclol precursors of the ergopeptines, has been partially purified and shown to consist of two polypeptide chains of 370 kDa (LPS 1) and 140 kDa (LPS 2); these contain all the sites necessary for the assembly of the D-lysergyl peptide backbone. The mechanism of D-lysergyl peptide synthesis remained unclear, however. RESULTS: We have identified the obligatory peptidic intermediates in D-lysergyl peptide synthesis and the sequential order of their formation. The two LPS subunits catalyze the formation of D-lysergyl mono-, di-, and tripeptides as enzyme-thioester intermediates, the formation of which appears to be irreversible. Peptide synthesis starts when D-lysergic acid binds to the LPS 2 subunit, which most probably occurs after the previous round of synthesis has been completed by the release of the end product from the LPS enzyme. CONCLUSIONS: We have shown that the mechanism of D-lysergyl peptide synthesis is an ordered process of successive acyl transfers on a multienzyme complex. This knowledge opens the way for enzymatic and genetic investigations into the formation of novel alkaloidcyclopeptides.
Authors: D G Panaccione; R D Johnson; J Wang; C A Young; P Damrongkool; B Scott; C L Schardl Journal: Proc Natl Acad Sci U S A Date: 2001-10-09 Impact factor: 11.205
Authors: Damien J Fleetwood; Barry Scott; Geoffrey A Lane; Aiko Tanaka; Richard D Johnson Journal: Appl Environ Microbiol Date: 2007-02-16 Impact factor: 4.792