Epimerization at C‐3′′ in Butirosin Biosynthesis by an NAD+‐Dependent Dehydrogenase BtrE and an NADPH‐Dependent Reductase BtrF

Abstract Butirosin is an aminoglycoside antibiotic consisting two epimers at C‐3′′ of ribostamycin/xylostasin with a unique 4‐amino‐2‐hydroxybutyrate moiety at C‐1 of the aminocyclitol 2‐deoxystreptamine (2DOS). To date, most of the enzymes encoded in the biosynthetic gene cluster for butirosin, from the producing strain Bacillus circulans , have been characterized. A few unknown functional proteins, including nicotinamide adenine dinucleotide cofactor‐dependent dehydrogenase/reductase (BtrE and BtrF), are supposed to be involved in the epimerization at C‐3′′ of butirosin B/ribostamycin but remain to be characterized. Herein, the conversion of ribostamycin to xylsostasin by BtrE and BtrF in the presence of NAD + and NADPH was demonstrated. BtrE oxidized the C‐3′′ of ribostamycin with NAD + to yield 3′′‐oxoribostamycin. BtrF then reduced the generated 3′′‐oxoribostamycin with NADPH to produce xylostasin. This reaction step was the last piece of butirosin biosynthesis to be described.