Characterization of a C3 Deoxygenation Pathway Reveals a Key Branch Point in Aminoglycoside Biosynthesis

Abstract

Apramycin is a clinically interesting aminoglycoside antibiotic (AGA) containing a highly unique bicyclic octose moiety, and this octose is deoxygenated at the C3 position. Although the biosynthetic pathways for most 2-deoxystreptamine-containing AGAs have been well characterized, the pathway for apramycin biosynthesis, including the C3 deoxygenation process, has long remained unknown. Here we report detailed investigation of apramycin biosynthesis by a series of genetic, biochemical and bioinformatical studies. We show that AprD4 is a novel radical S-adenosyl-l-methionine (SAM) enzyme, which uses a noncanonical CX3CX3C motif for binding of a [4Fe-4S] cluster and catalyzes the dehydration of paromamine, a pseudodisaccharide intermediate in apramycin biosynthesis. We also show that AprD3 is an NADPH-dependent reductase that catalyzes the reduction of the dehydrated product from AprD4-catalyzed reaction to generate lividamine, a C3' deoxygenated product of paromamine. AprD4 and AprD3 do not form a tight catalytic complex, as shown by protein complex immunoprecipitation and other assays. The AprD4/AprD3 enzyme system acts on different pseudodisaccharide substrates but does not catalyze the deoxygenation of oxyapramycin, an apramycin analogue containing a C3 hydroxyl group on the octose moiety, suggesting that oxyapramycin and apramycin are partitioned into two parallel pathways at an early biosynthetic stage. Functional dissection of the C6 dehydrogenase AprQ shows the crosstalk between different AGA biosynthetic gene clusters from the apramycin producer Streptomyces tenebrarius, and reveals the remarkable catalytic versatility of AprQ. Our study highlights the intriguing chemistry in apramycin biosynthesis and nature's ingenuity in combinatorial biosynthesis of natural products.

Parallel pathways in the biosynthesis of aminoglycoside antibiotics

“…In contrast to the previous proposal in which the C3 deoxygenation occurs as the penultimate step, biochemical studies have shown that the C3 deoxygenation is catalyzed by the radical SAM diol dehydratase AprD4 and its NADPH-dependent reductase partner AprD3 20, 39, 40 . This C3 deoxygenation reaction does not occur on oxyapramycin but on the pseudodisaccharide substrate paromamine, demonstrating the two parallel biosynthetic pathways, which affords oxyapramycin and apramycin, respectively ( Figure 3) 20 . Consistent with this observation, biochemical analysis has shown that AprQ, a FAD-dependent dehydrogenase, catalyzes the C6' dehydrogenation of both paromamine and lividamine, with the latter as the preferred substrate 20 .…”

“…Consistent with this observation, biochemical analysis has shown that AprQ, a FAD-dependent dehydrogenase, catalyzes the C6' dehydrogenation of both paromamine and lividamine, with the latter as the preferred substrate 20 . Remarkably, AprQ is also able to act on the tripseudodisaccharide substrate gentamicin X2 and G418 and, in the latter case, produce a novel gentamicin analogue with a C6' carboxylate 20 . A similar observation was also made on AprD4 and AprD3, which are able to act on tripseudodisaccharide substrates, such as kanamycin C and kanamycin B 40 .…”

“…This C3 deoxygenation reaction does not occur on oxyapramycin but on the pseudodisaccharide substrate paromamine, demonstrating the two parallel biosynthetic pathways, which affords oxyapramycin and apramycin, respectively ( Figure 3) 20 . Consistent with this observation, biochemical analysis has shown that AprQ, a FAD-dependent dehydrogenase, catalyzes the C6' dehydrogenation of both paromamine and lividamine, with the latter as the preferred substrate 20 . Remarkably, AprQ is also able to act on the tripseudodisaccharide substrate gentamicin X2 and G418 and, in the latter case, produce a novel gentamicin analogue with a C6' carboxylate 20 .…”

CRISPR-Cas9/Cas12a biotechnology and application in bacteria

“…Streptomyces is the largest genus of Actinobacteria with an extremely high GC content in their genomes. Streptomyces have evolved to produce a wide variety of bioactive secondary metabolites, including antibiotics, anticancer agents, herbicides, and immunosuppressants [ [154] , [155] , [156] , [157] , [158] , [159] , [160] , [161] , [162] , [163] , [164] , [165] , [166] ]. However, the potential for metabolite biosynthesis is not fully explored.…”

Confronting the catalytic dark matter encoded by sequenced genomes

“…These cell culture-based findings were corroborated with observations from a whole-organism model when tissue extracts of homozygous ABHD3 knockout mice were compared with extracts of WT mice, and C14-lysophosphatidylcholine was found to accumulate in addition to three other phosphatidylcholines ( 74 ). Ex-vivo metabolomics profiling also led to the discovery and characterization of sedoheptulose-1,7-bisphosphatase, which is involved in riboneogenesis in yeast ( 75 ), a radical SAM dehydratase as well as an NADPH-dependent reductase involved in the biosynthesis of the aminoglycoside antibiotic apramycin in Streptomyces tenebrarius ( 76 ), and the yeast DLD3 protein as a D-2-hydroxyglutarate-pyruvate transhydrogenase potentially involved in the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial electron transfer chain ( 77 ). Most recently, the human FGGY protein and its yeast ortholog were also identified as D-ribulokinases using this experimental approach as a starting point ( 78 ).…”