Chapter Five - The Pictet–Spengler Mechanism Involved in the Biosynthesis of Tetrahydroisoquinoline Antitumor Antibiotics: A Novel Function for a Nonribosomal Peptide Synthetase

Abstract

The core scaffold of microbial tetrahydroisoquinoline antitumor antibiotics is biosynthesized by a nonribosomal peptide synthetase (NRPS) with novel functions, which catalyzes a highly unusual seven-step transformation involving multiple reductions of thioester intermediates and two rounds of the Pictet-Spengler reaction. The reaction mechanism of saframycin NRPS SfmC has been firmly established by a series of in vitro experiments using various substrate analogs, SfmC domain-deletion mutants and (2)H-labeled NADH and NADPH. The Pictet-Spengler reaction found in the biosynthesis of saframycin heavily relies on the chain length of the cryptic long acyl chain in the peptide substrates. This chapter describes protocols for biochemical characterization of the saframycin NRPS SfmC. They include (1) bioinformatic analysis of related gene clusters, (2) synthesis of intermediate analogs, and (3) enzymatic reactions for both analytical and preparative scale.