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′-²H]-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.

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′-²H]-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.

DesII is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the C4-deamination of TDP-4-amino-4,6-dideoxyglucose through a C3 radical intermediate. However, if the C4 amino group is replaced with a hydroxy group (to give TDP-quinovose), the hydroxy group at C3 is oxidized to a ketone with no C4-dehydration. It is hypothesized that hyperconjugation between the C4 CN/O bond and the partially filled p orbital at C3 of the radical intermediate modulates the degree to which elimination competes with dehydrogenation. To investigate this hypothesis, the reaction of DesII with the C4-epimer of TDP-quinovose (TDP-fucose) was examined. The reaction primarily results in the formation of TDP-6-deoxygulose and likely regeneration of TDP-fucose. The remainder of the substrate radical partitions roughly equally between C3-dehydrogenation and C4-dehydration. Thus, changing the stereochemistry at C4 permits a more balanced competition between elimination and dehydrogenation.

DesII is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the C4-deamination of TDP-4-amino-4,6-dideoxyglucose through a C3 radical intermediate. However, if the C4 amino group is replaced with a hydroxy group (to give TDP-quinovose), the hydroxy group at C3 is oxidized to a ketone with no C4-dehydration. It is hypothesized that hyperconjugation between the C4 CN/O bond and the partially filled p orbital at C3 of the radical intermediate modulates the degree to which elimination competes with dehydrogenation. To investigate this hypothesis, the reaction of DesII with the C4-epimer of TDP-quinovose (TDP-fucose) was examined. The reaction primarily results in the formation of TDP-6-deoxygulose and likely regeneration of TDP-fucose. The remainder of the substrate radical partitions roughly equally between C3-dehydrogenation and C4-dehydration. Thus, changing the stereochemistry at C4 permits a more balanced competition between elimination and dehydrogenation.

Following the biosynthesis of polyketide backbones by polyketide synthases (PKSs), post-PKS modifications result in a significantly elevated level of structural complexity that renders the chemical synthesis of these natural products challenging. We report herein a total synthesis of the widely used polyketide insecticide spinosyn A by exploiting the prowess of both chemical and enzymatic methods. As more polyketide biosynthetic pathways are characterized, this chemoenzymatic approach is expected to become readily adaptable to streamlining the synthesis of other complex polyketides with more elaborate post-PKS modifications.

Following the biosynthesis of polyketide backbones by polyketide synthases (PKSs), post-PKS modifications result in a significantly elevated level of structural complexity that renders the chemical synthesis of these natural products challenging. We report herein a total synthesis of the widely used polyketide insecticide spinosyn A by exploiting the prowess of both chemical and enzymatic methods. As more polyketide biosynthetic pathways are characterized, this chemoenzymatic approach is expected to become readily adaptable to streamlining the synthesis of other complex polyketides with more elaborate post-PKS modifications.

Many biologically active small-molecule natural products produced by microorganisms derive their activities from sugar substituents. Changing the structures of these sugars can have a profound impact on the biological properties of the parent compounds. This realization has inspired attempts to derivatize the sugar moieties of these natural products through exploitation of the sugar biosynthetic machinery. This approach requires an understanding of the biosynthetic pathway of each target sugar and detailed mechanistic knowledge of the key enzymes. Scientists have begun to unravel the biosynthetic logic behind the assembly of many glycosylated natural products and have found that a core set of enzyme activities is mixed and matched to synthesize the diverse sugar structures observed in nature. Remarkably, many of these sugar biosynthetic enzymes and glycosyltransferases also exhibit relaxed substrate specificity. The promiscuity of these enzymes has prompted efforts to modify the sugar structures and alter the glycosylation patterns of natural products through metabolic pathway engineering and enzymatic glycodiversification. In applied biomedical research, these studies will enable the development of new glycosylation tools and generate novel glycoforms of secondary metabolites with useful biological activity.

Viele biologisch aktive niedermolekulare Naturstoffe, die von Mikroorganismen produziert werden, leiten ihre Aktivitäten von Zuckersubstituenten ab. Eine Veränderung der Struktur dieser Zucker kann starke Auswirkungen auf die biologischen Eigenschaften der Stammverbindungen haben. Diese Erkenntnis hat zu Bestrebungen geführt, die Zuckerreste dieser Naturstoffe mithilfe des Zuckerbiosyntheseapparats zu derivatisieren. Hierfür müssen die jeweiligen Biosynthesewege und die Mechanismen der Schlüsselenzyme im Detail bekannt sein. In der Biochemie beginnt man allmählich, die Biosyntheseprinzipien, die dem Aufbau vieler glycosylierter Naturstoffe zugrunde liegen, zu verstehen, und man konnte einen Satz von Enzymaktivitäten ausmachen, die für die Synthese der vielfältigen in der Natur beobachteten Zuckerstrukturen zuständig sind. Bemerkenswerterweise zeigen viele dieser Zuckerbiosyntheseenzyme ebenso wie Glycosyltransferasen eine relaxierte Substratspezifität. Die Promiskuität dieser Enzyme führte zu Bestrebungen, die Zuckerstrukturen zu modifizieren und die Glycosylierungsmuster von Naturstoffen durch Planung der Stoffwechselwege oder durch enzymatische Glycodiversifizierung zu verändern. In der praktischen biomedizinischen Forschung werden diese Studien zur Entwicklung neuer Glycosylierungsmittel führen und bei Sekundärmetaboliten neue Glycoformen mit nützlichen biologischen Aktivitäten hervorbringen.