“…Chen et al have determined the enzymatic mechanism of C-5 methylation for the nucleoside skeleton and further solved the crystal structure of the C-5 methylase, PolB (12). Previous works also initiated the production of polyoxin-nikkomycin (POL-NIK) hybrid nucleoside antibiotics through synthetic biology approaches (13)(14)(15).…”
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.
“…Chen et al have determined the enzymatic mechanism of C-5 methylation for the nucleoside skeleton and further solved the crystal structure of the C-5 methylase, PolB (12). Previous works also initiated the production of polyoxin-nikkomycin (POL-NIK) hybrid nucleoside antibiotics through synthetic biology approaches (13)(14)(15).…”
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.
“…Two of the hybrid antibiotics, polyoxin N and nikkoxin D, were significantly more potent against human or plant fungal pathogens. It may be used for generation of novel peptidyl nucleoside antibiotics in industrial strains (Zhai et al 2012).…”
Number of microorganisms produces antibiotics that can inhibit or kill the other microbes. The production of some antibiotics is not sufficient in native host rather difficult to synthesize chemically and to extract in large amounts for commercialization. Metabolic engineering plays an increasingly significant role in the production of antibiotics and its precursors. Thus, we engineer biosynthetic pathways in desire host for the production of sufficient quantity of antibiotics. In this chapter, we illustrated bioengineering of different microbes using synthetic biology and metabolic engineering approaches for production and regulation of antibiotics.
“…1B) (Chen et al, 2009). The C-5 modifications within the nucleoside skeleton confer not only structural diversity but also possess a bioactivity preference for polyoxin (Isono et al, 1967; Isono and Suzuki, 1968; Isono et al, 1975; Zhai et al, 2012). Previous labeling studies indicated that the C-5 methylation originated from C-3 of serine and is catalyzed by a new enzyme independent of thymidylate synthase (Isono and Suhadolnik, 1976; Isono, 1988), however, the molecular mechanism for such modification remained elusive for decades.Figure 1
Gene cluster and structure of polyoxin as well as the dual functions of PolB .…”
Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate synthase activity which is responsible for the C-5 methylation of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 Å, 1.76 Å and 2.28 Å resolutions, respectively. Loop 1 (residues 117–131), Loop 2 (residues 192–201) and the substrate recognition peptide (residues 94–102) of PolB exhibit considerable conformational flexibility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methylase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-016-0289-y) contains supplementary material, which is available to authorized users.
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