Nine Enzymes Are Required for Assembly of the Pacidamycin Group of Peptidyl Nucleoside Antibiotics

Abstract

Pacidamycins are a family of uridyl peptide antibiotics that inhibit the translocase MraY, an essential enzyme in bacterial cell wall biosynthesis not yet clinically targeted. The pacidamycin structural skeleton contains a doubly inverted peptidyl chain with a β-peptide and a ureido linkage, and a 3’-deoxyuridine nucleoside attached to DABA3 of the peptidyl chain via an enamide linkage. Although the biosynthetic gene cluster for pacidamycins was identified recently, the assembly line of this group of peptidyl nucleoside antibiotics remained poorly understood due to the highly dissociated nature of the encoded nonribosomal peptide synthetase (NRPS) domains and modules. In this work, a minimum set of enzymes were identified in generating the pacidamycin scaffold from amino acid and nucleoside monomers, highlighting a freestanding thiolation (T) domain (PacH) as a key carrier component in the peptidyl chain assembly, as well as a freestanding condensation (C) domain (PacI) catalyzing the release of the assembled peptide by a nucleoside moiety. Based on the substrate promiscuity of this enzymatic assembly line, several pacidamycin analogs were produced using in vitro total biosynthesis.

tRNA-dependent peptide bond formation by the transferase PacB in biosynthesis of the pacidamycin group of pentapeptidyl nucleoside antibiotics

“…S2). We have previously shown that uridyl tetrapeptides with N-terminal m-Tyr tethered to the β-amino of DABA could be formed using the nine NRPS and associated enzymes PacDHIJLNOPW in vitro (9). No uridyl pentapeptide could be detected from those enzymatic reactions.…”

“…Bioinformatic analysis revealed the building blocks in pacidamycins are assembled using a nonribosomal peptide synthetase (NRPS) thiotemplate logic, whereas the encoded NRPS modules and domains are highly dissociated and predicted to work with each other in trans. We further characterized the functions of the separate NRPS protein domains in vitro to delineate catalytic steps for activation and insertion of each building block in the pacidamycin scaffold (8,9). We have shown that nine NRPS enzymes encoded by the producing organism S. coeruleorubidus, when heterologously expressed and purified from Escherichia coli, allowed reconstitution of the 5′-aminouridyl-tetrapeptide framework of the pacidamycin family (Fig.…”

“…No putative C domain was found to be involved in the attachment of L-Ala/L-m-Tyr to the β-amino of DABA after activation by the dedicated adenylation (A) domains (PacU/PacW) ( Fig. 1) (9). As the functions of all of the NRPS enzymes encoded by the gene cluster have been elucidated, the molecular mechanism of the activation and insertion of the N-terminal L-Ala/L-Gly in the pacidamycin pentapeptidyl scaffold may therefore involve an NRPS-unrelated mechanism.…”

Simamycin (5′-O-geranyluridine): a new prenylated nucleoside from Streptomyces sp.

“…The cis-diol of uridine was protected as dimethylacetonide, 8 which was then O-alkylated using geranyl bromide in the presence of NaH to give 4. 9 Geranylation at the 5-position of ribose was confirmed by an HMBC correlation from H-12 to C-11.…”

Inhibition of phospho-MurNAc-pentapeptide translocase (MraY) by nucleoside natural product antibiotics, bacteriophage ϕX174 lysis protein E, and cationic antibacterial peptides

“…Two new series of peptidyl-uridines whose structures are based upon mureidomycin A and tunicamycin have been synthesised as inhibitors of C. jejuni PglC, and selected compounds show IC50 values in the range 40-250 µM [35]. The biosynthetic pathway to both the pacidamycin [36][37][38] and caprazamycin [39] antibiotics has been elucidated, hence the use of biosynthetic engineering offers the possibility of generating modified uridyl peptide antibiotics via fermentation in the future. shown that mureidomycin A and liposidomycin B are both slow-binding inhibitors, while tunicamycin is a reversible inhibitor [40,41].…”