Enzymatic N- and C-Protection in Cyanobactin RiPP Natural Products

Abstract

Recent innovations in peptide natural product biosynthesis reveal a surprising wealth of previously uncharacterized biochemical reactions that have potential applications in synthetic biology. Among these, the cyanobactins are noteworthy because these peptides are protected at their N- and C-termini by macrocyclization. Here, we use a novel bifunctional enzyme AgeMTPT to protect linear peptides by attaching prenyl and methyl groups at their free N- and C-termini. Using this peptide protectase in combination with other modular biosynthetic enzymes, we describe the total synthesis of the natural product aeruginosamide B and the biosynthesis of linear cyanobactin natural products. Our studies help to define the enzymatic mechanism of macrocyclization, providing evidence against the water exclusion hypothesis of transpeptidation and favoring the kinetic lability hypothesis.

Post-Translational Tyrosine Geranylation in Cyanobactin Biosynthesis

“…Although for many cyanobactins the substrate is cyclic, in the case of linear cyanobactins aeruginosamides, the prenyltransferase adds DMAPP to the N-terminus prior to the action of a PatG-like protease. 18 To compare the two substrates, kinetic parameters were measured with cyc-MSGVDYYNP ( 2 ) and MSGVDYYNPFAGDDAE ( 4 ) (Table 2, Figure S2). Kinetic values were very similar for both substrates, being identical within error.…”

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

“…While each prenyltransferase appears to be relatively selective for the amino acid, orientation, and isoprene donor, there is much less selectivity as far as peptide structure, and no known associated RS (Hao et al, 2016; McIntosh et al, 2011). One variant homolog carries out prenylation of the N-terminus, rather than the side chain, on peptide substrates (Leikoski et al, 2013; Sardar et al, 2017). …”

“…Methyltransferases are also very common within the genetic organization of cyanobactin pathways, but thus far only one has been characterized (Donia & Schmidt, 2011; Leikoski et al, 2009; Leikoski et al, 2013; Sardar et al, 2017). …”

“…Another divergent pathway is that involved in the biosynthesis of linear cyanobactins, such as aeruginosamides (Leikoski et al, 2013; Sardar et al, 2017). In the age pathway, the PatG homolog AgeG produced a linear peptide product, rather than performing macrocyclization.…”

“…The major exception to this rule is the aeruginosamide ( age ) pathway, where AgeG performs C-terminal hydrolysis to liberate a linear product, in place of macrocyclization (Leikoski et al, 2013; Sardar et al, 2017). …”