AprD4, the only characterised protein in this family as of June 2016, is the first known radical SAM diol-dehydratase and, along with AprD3, is responsible for 3'-deoxygenation in aminoglycoside biosynthesis.
Kim HJ, LeVieux J, Yeh YC, Liu HW
C3'-Deoxygenation of Paromamine Catalyzed by a Radical S-Adenosylmethionine Enzyme: Characterization of the Enzyme AprD4 and its Reductase Partner AprD3
▸ Abstract
C3'-deoxygenation of aminoglycosides results in their decreased susceptibility to phosphorylation thereby increasing their efficacy as antibiotics. However, the biosynthetic mechanism of C3'-deoxygenation is unknown. To address this issue, aprD4 and aprD3 genes from the apramycin gene cluster in Streptomyces tenebrarius were expressed in E. coli and the resulting gene products were characterized in vitro. AprD4 is shown to be a radical S-adenosylmethionine (SAM) enzyme, catalyzing homolysis of SAM to 5'-deoxyadenosine (5'-dAdo) in the presence of paromamine. [4'-(2) H]-Paromamine was prepared and used to show that its C4'-H is transferred to 5'-dAdo by AprD4, during which the substrate is dehydrated to a product consistent with 4'-oxolividamine. In contrast, paromamine is reduced to a deoxy product when incubated with AprD4/AprD3/NADPH. These results show that AprD4 is the first radical SAM diol-dehydratase and, along with AprD3, is responsible for 3'-deoxygenation in aminoglycoside biosynthesis.
Angew Chem Int Ed Engl
2016;None(None):None-None
| PubMed ID:
26879038
Lv M, Ji X, Zhao J, Li Y, Zhang C, Su L, Ding W, Deng Z, Yu Y, Zhang Q
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 non-canonical 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 analog 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.
J Am Chem Soc
2016;None(None):None-None
| PubMed ID:
27120352
Members of this family have a non-canonical CX3CX3C motif for binding of a [4Fe-4S] cluster. They catalyse the dehydration of paromamine, a pseudodisaccharide intermediate in apramycin biosynthesis.
