Bypassing the proline/thiazoline requirement of the macrocyclase PatG

Abstract

Macrocyclisation of fully non-peptidic compounds and non-heterocycle containing macrocycles using the peptidic ligase PatGmac.

The Biochemistry and Structural Biology of Cyanobactin Pathways: Enabling Combinatorial Biosynthesis

“…Even so, PatG accepts substrates with proline substituting for thiazoline, as well as triazoles and, interestingly, heterocycles comprised of disulfide linked cysteine-cysteine (e.g., CC-SYD, etc.) (Oueis et al, 2016; Oueis, Stevenson, et al, 2017). These are synthetically useful alternatives in the creation of large molecules.…”

“…The minimal substrate requirement for macrocyclization by PatG (i.e. minimal three residue follower sequence preceded by a heterocycle) has prompted several efforts focused on utilizing the isolated protease domain as a biotechnology tool (Alexandru-Crivac et al, 2017; McIntosh, Robertson, et al, 2010; Oueis et al, 2016; Oueis, Nardone, et al, 2017; Oueis, Stevenson, et al, 2017; Sardar, Lin, et al, 2015). For example, the macrocyclase was predicted to be useful in producing discrete compounds such as peptides that mimic hormones or neuropeptides, or libraries of natural unnatural products.…”

“…The P2 position is also somewhat fastidious, in that substrates containing D-amino acids or glycine in that position were not substrates for PatG (McIntosh, Robertson, et al, 2010). A later study suggested that the L-amino acid is critical for correct positioning of the peptide in the active site (Oueis, Stevenson, et al, 2017). …”

“…PatG and related proteases cleave before RSIII (Figure 9). Both PatG and TruG are capable of recognizing the C-terminal sequences X-AYD-COOH, X-SYD-COOH, X-AYDG-COOH, X-AYDGE-COOH, X-SYDD-COOH, and relatives (X- indicates the position of cleavage) (Lee et al, 2009; McIntosh, Robertson, et al, 2010; Oueis et al, 2015; Oueis, Jaspars, Westwood, & Naismith, 2016; Oueis, Stevenson, Jaspars, Westwood, & Naismith, 2017; Sardar, Lin, et al, 2015). In addition, they recognize slightly longer sequences resulting from cleavage of multiple cassettes, such as X-AYDGLEAS-COOH (Lee et al, 2009).…”

Expanding the chemical space of synthetic cyclic peptides using a promiscuous macrocyclase from prenylagaramide biosynthesis

“…While proline and other heterocycles in artificial substrates is sometimes accepted by PatG, the reaction is usually much slower than it is with azoline. [30][31] Generally, azolines are installed by a specific cyanobactin heterocyclase enzyme prior to macrocyclization. 32 Although the heterocycle provides rigidity to the backbone to assist macrocyclization, this additional biochemical step increases complexity and compromises the yield for downstream applications such as library generation.…”