关键词: S-腺苷甲硫氨酸自由基酶, 三残基环番合成酶, 核糖体肽, 翻译后修饰

Abstract:

Cyclic ribosomally synthesized and post-translationally modified peptides (RiPPs) with macrocycles derived from three amino acid residues have attracted intense interest in recent years. Biosynthesis of these peptides usually involves a radical (S)-adenosylmethionine (rSAM) enzyme that catalyzes the formation of a C—C or C—O bond between an aromatic carbon and a side chain carbon of another residue. Such enzymes are named three-residue cyclophane forming enzymes (3-CyFEs). The rSAM enzyme family is known as one of the largest enzyme super families, which consists of more than 22000 members. rSAM enzymes are widely found in all three life domains as one of the earliest biocatalysts on earth. A large amount of microbial genomic information shows that many RiPPs biosynthesis gene clusters contain rSAM enzymes. Recently, several 3-CyFEs from Xye gene clusters, which form three cyclizations on the precursor peptides, were reported. In this paper, we report a new Xye RiPP pacpeptide and its key biosynthetic enzyme PacB from Photorhabdus australis DSM 17609. We coexpressed the pacA and pacB in Escherichia coli and reconstituted the activity of heterologously expressed protein to investigate the bioactivity of 3-CyFEs PacB. The high-resolution mass spectrometry assay indicates that PacB indeed led to forming all three cyclophanes of pacpeptide. Additionally, the mutation of Asn/Argto Gly on the precursor peptide resulted in only two cyclophane was found, which demonstrated that three cyclization reactions were generated independently with different efficiency. PacB also catalyzed all three cyclophanes formation on the precursor peptide with Asn/Arg mutated to Ala. These results expand our understanding of 3-CyFEs chemistry in natural product biosynthesis and provide insights into the post-modification of cyclic RiPPs with three-residue cyclophanes.

Key words: rSAM enzyme, 3-CyFEs, RiPPs, post-translational modification