An ATP-Dependent Ligase with Substrate Flexibility Involved in Assembly of the Peptidyl Nucleoside Antibiotic Polyoxin

Abstract

Polyoxin (POL) is an unusual peptidyl nucleoside antibiotic, in which the peptidyl moiety and nucleoside skeleton are linked by an amide bond. However, their biosynthesis remains poorly understood. Here, we report the deciphering of PolG as an ATP-dependent ligase responsible for the assembly of POL. A mutant is capable of accumulating multiple intermediates, including the peptidyl moiety (carbamoylpolyoxamic acid [CPOAA]) and the nucleoside skeletons (POL-C and the previously overlooked thymine POL-C). We further demonstrate that PolG employs an ATP-dependent mechanism for amide bond formation and that the generation of the hybrid nucleoside antibiotic POL-N is also governed by PolG. Finally, we determined that the deduced ATP-binding sites are functionally essential for PolG and that they are highly conserved in a number of related ATP-dependent ligases. These insights have allowed us to propose a catalytic mechanism for the assembly of peptidyl nucleoside antibiotic via an acyl-phosphate intermediate and have opened the way for the combinatorial biosynthesis/pathway engineering of this group of nucleoside antibiotics. POL is well known for its remarkable antifungal bioactivities and unusual structural features. Actually, elucidation of the POL assembly logic not only provides the enzymatic basis for further biosynthetic understanding of related peptidyl nucleoside antibiotics but also contributes to the rational generation of more hybrid nucleoside antibiotics via synthetic biology strategy.

Conserved Mechanism of 2′-Phosphorylation-Aided Amide Ligation in Peptidyl Nucleoside Biosynthesis

“…When PolG was assayed with either AHAP or 5cAHA, PolG ligated both 5cAHA and AHAP with CPOAA and produced polyoxin D and polyoxin L 2′-phosphate, respectively, in an ATP-dependent fashion (Figure A). The observed activity of PolG with 5cAHA is consistent with the previous report . However, when the PolG assay was performed with stoichiometric amounts (100 μM) of AHAP, 5cAHA, and CPOAA, AHAP was completely consumed and converted into polyoxin L 2′-phosphate, while 5cAHA remained mostly unreacted with little to no formation of polyoxin D (Figure A, trace iii).…”

“…Initially, we qualitatively assessed PolG’s specificity among 5cAHA, AHA, and AHAP. PolG was expressed and purified as an N-terminally His 6 -tagged protein as described previously . All PolG assays were performed using CPOAA as the carboxylate substrate.…”

“…In the PN biosynthesis, dephosphorylation timing remains ambiguous due to conflicting observations about the pathway's last step, formation of the amide bond between AHA and the Nterminal amino acid. While the early characterization of PolG demonstrated its in vitro activity to ligate nonphosphorylated AHA and CPOAA in an ATP-dependent manner (Figure 1A), 11 recent characterization of NikS demonstrated its activity with AHA 2′-phosphate [AHAP (6) (Figure 1B)]. 6 These PolG and NikS characterizations were qualitative without kinetic studies, and the activity of PolG has never been studied with 2′-phosphorylated nucleosides.…”

“…PolG was expressed and purified as an N-terminally His 6tagged protein as described previously. 11 All PolG assays were performed using CPOAA as the carboxylate substrate. CPOAA was prepared by acid hydrolysis of polyoxin D followed by HPLC purification.…”

Cryptic phosphorylation in nucleoside natural product biosynthesis

“…Therefore, AHAP is likely the physiological substrate of NikS, and AHA is an off-pathway shunt metabolite. Intriguingly, in a recent report for PolG 42 , the NikS homolog in the polyoxin pathway, recombinant PolG ligates AHA and CPOAA ( Figure 1a ). Together with our studies, this earlier report suggests that PolG may promiscuously accept both AHA and AHAP, while only AHAP is physiologically relevant.…”