C3'-deoxygenation of aminoglycosides results in their decreased susceptibility to phosphorylation thereby increasing their efficacy as antibiotics.H owever,t he 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 aradical S-adenosylmethionine (SAM) enzyme,c atalyzing homolysis of SAM to 5'-deoxyadenosine (5'-dAdo) in the presence of paromamine.[ 4 '-2

Oxetanocin A and albucidin are two oxetane natural products. While the biosynthesis of oxetanocin A has been described, less is known about albucidin. In this work, the albucidin biosynthetic gene cluster is identified in Streptomyces. Heterologous expression in a nonproducing strain demonstrates that the genes alsA and alsB are necessary and sufficient for albucidin biosynthesis confirming a previous study (Myronovskyi et al. Microorganisms 2020, 8, 237). A two-step construction of albucidin 4'-phosphate from 2'-deoxyadenosine monophosphate (2'-dAMP) is shown to be catalyzed in vitro by the cobalamin dependent radical Sadenosyl-L-methionine (SAM) enzyme AlsB, which catalyzes a ring contraction, and the radical SAM enzyme AlsA, which catalyzes elimination of a onecarbon fragment. Isotope labelling studies show that AlsB catalysis begins with stereospecific H-atom transfer of the C2'-pro-R hydrogen from 2'-dAMP to 5'deoxyadenosine, and that the eliminated one-carbon fragment originates from C3' of 2'-dAMP.

OxsB is a B12-dependent radical SAM enzyme that catalyzes the oxidative ring contraction of 2′-deoxyadenosine 5′-phosphate to the dehydrogenated, oxetane containing precursor of oxetanocin A phosphate. AlsB is a homologue of OxsB that participates in a similar reaction during the biosynthesis of albucidin. Herein, OxsB and AlsB are shown to also catalyze radical mediated, stereoselective C2′-methylation of 2′-deoxyadenosine monophosphate. This reaction proceeds with inversion of configuration such that the resulting product also possesses a C2′ hydrogen atom available for abstraction. However, in contrast to methylation, subsequent rounds of catalysis result in C–C dehydrogenation of the newly added methyl group to yield a 2′-methylidene followed by radical addition of a 5′-deoxyadenosyl moiety to produce a heterodimer. These observations expand the scope of reactions catalyzed by B12-dependent radical SAM enzymes and emphasize the susceptibility of radical intermediates to bifurcation along different reaction pathways even within the highly organized active site of an enzyme.

Oxetanocin A and albucidin are two oxetane natural products. While the biosynthesis of oxetanocin A has been described, less is known about albucidin. In this work, the albucidin biosynthetic gene cluster is identified in Streptomyces. Heterologous expression in a nonproducing strain demonstrates that the genes alsA and alsB are necessary and sufficient for albucidin biosynthesis confirming a previous study (Myronovskyi et al. Microorganisms 2020, 8, 237). A two‐step construction of albucidin 4′‐phosphate from 2′‐deoxyadenosine monophosphate (2′‐dAMP) is shown to be catalyzed in vitro by the cobalamin dependent radical S‐adenosyl‐l‐methionine (SAM) enzyme AlsB, which catalyzes a ring contraction, and the radical SAM enzyme AlsA, which catalyzes elimination of a one‐carbon fragment. Isotope labelling studies show that AlsB catalysis begins with stereospecific H‐atom transfer of the C2′‐pro‐R hydrogen from 2′‐dAMP to 5′‐deoxyadenosine, and that the eliminated one‐carbon fragment originates from C3′ of 2′‐dAMP.