Two Cryptic Self‐Resistance Mechanisms in Streptomyces tenebrarius Reveal Insights into the Biosynthesis of Apramycin

Abstract

Apramycin is aclinically promising aminoglycoside antibiotic (AGA). To date,m echanisms underlying the biosynthesis and self-resistance of apramycin remain largely unknown. Here we report that apramycin biosynthesis proceeds through unexpected phosphorylation, deacetylation, and dephosphorylation steps,i nw hich an ovel aminoglycoside phosphotransferase (AprU), ap utative creatinine amidohydrolase (AprP), and an alkaline phosphatase (AprZ) are involved. Biochemical characterization revealed that AprU specifically phosphorylates 5-OH of ap seudotrisaccharide intermediate,w hose N-7' acetyl group is subsequently hydrolyzed by AprP.A prZ is located extracellularly where it removes the phosphate group from ap seudotetrasaccharide intermediate,leading to the maturation of apramycin. Intriguingly,7'-N-acetylated and 5-O-phosphorylated apramycin that were accumulated in DaprU and DaprZ respectively exhibited significantly reduced antibacterial activities,i mplying Streptomyces tenebrarius employs C-5 phosphorylation and N-7' acetylation as two strategies to avoid auto-toxicity.S ignificantly,t his study provides insight into the design of new generation AGAs to circumvent the emergence of drugresistant pathogens.

Biosynthetic Origin of the Octose Core and Its Mechanism of Assembly during Apramycin Biosynthesis

“…A significant amount of the carboxylic acid 18 was also generated with GalNAc ( 16 ) as the C 2 -donor in the one-pot reaction (Figure B, trace b, Figure S7) indicating that 6′-oxolividamine ( 6 ) was not fully consumed by AprG but was instead further oxidized by BtrQ to 18 . Therefore, AprU, which catalyzes 5-phosphorylation of 7 , and ATP were included in the BtrQ/AprG reaction in order to drive the AprG-catalyzed reaction toward the phosphorylated octose product 8 . LC-MS analysis showed that the signal corresponding to 18 (calcd C 12 H 23 N 3 O 7 [M + H] + 322.1609) was no longer discernible being replaced by a signal at m / z 487.1789 consistent with accumulation of 8 (calcd C 16 H 31 N 4 O 11 P [M + H] + 487.1800, Figure B, trace c).…”

“…While LC-MS analysis of the BtrQ/AprG/AprU reaction product described above was consistent with the formation of 7 and thus AprG-catalyzed octose formation, the structure of this product remained fully established. Therefore, AprZ (fused with maltose-binding protein at N-terminal (MBP-AprZ)), which is an alkaline phosphatase that catalyzes C5-dephosphorylation of apramycin-5-phosphate ( 11 ) and its biosynthetic intermediates, was used to convert the isolated BtrQ/AprG/AprU reaction product ( 8 ) back to 7 . As shown in Figure S9, dephosphorylation of the product catalyzed by AprZ was indeed observed, indicating that the inclusion of AprU in the initial reaction followed by workup with AprZ helped to improve production of 7 .…”

“…How apramycin ( 1 ) is assembled has attracted much attention over the years. − The identification of its biosynthetic gene cluster ( apr cluster) − lead to significant progress in understanding the biological formation of apramycin (see Figure ). Apramycin ( 1 ) contains five amino groups at C1, C3, C2′, C7′ and C4″.…”

“…Biosynthetic pathway for apramycin (1). 17,21,24,25 Conversion of paromamine (4) to lividamine (5) is catalyzed by AprD4 and AprD3. 17−19 Subsequent AprQ mediated C6′ oxidation of 5 to oxolividamine (6) 17 sets the stage for octose assembly.…”

From solo to duet, intersections of natural product assembly with self-resistance

“…3D). 22 The self-resistance strategy involving a prodrug is widely recognized to apply a protective group that needs to be removed for the molecule to exhibit bioactivity. 11,23 A protective group could be installed onto the NP scaffold at different stages.…”