Characterization and Mechanistic Study of a Radical SAM Dehydrogenase in the Biosynthesis of Butirosin

BtrN encoded in the butirosin biosynthetic gene cluster possesses a CXXXCXXC motif conserved within the radical S-adenosyl methionine (SAM) superfamily. Its gene disruption in the butirosin producer Bacillus circulans caused the interruption of the biosynthetic pathway between 2-deoxy-scyllo-inosami...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2007-12, Vol.129 (49), p.15147-15155
Main Authors: Yokoyama, Kenichi, Numakura, Mario, Kudo, Fumitaka, Ohmori, Daijiro, Eguchi, Tadashi
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Anti-Bacterial Agents - biosynthesis
Bacillus - enzymology
Bacillus - genetics
Bacillus - metabolism
Butirosin Sulfate - biosynthesis
Hexosamines - metabolism
Kinetics
Mutagenesis, Insertional
Oxidoreductases - genetics
Oxidoreductases - metabolism
S-Adenosylmethionine - metabolism
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Summary:BtrN encoded in the butirosin biosynthetic gene cluster possesses a CXXXCXXC motif conserved within the radical S-adenosyl methionine (SAM) superfamily. Its gene disruption in the butirosin producer Bacillus circulans caused the interruption of the biosynthetic pathway between 2-deoxy-scyllo-inosamine (DOIA) and 2-deoxystreptamine (DOS). Further, in vitro assay of the overexpressed enzyme revealed that BtrN catalyzed the oxidation of DOIA under the strictly anaerobic conditions along with consumption of an equimolar amount of SAM to produce 5‘-deoxyadenosine, methionine, and 3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI). Kinetic analysis showed substrate inhibition by DOIA but not by SAM, which suggests that the reaction is the Ordered Bi Ter mechanism and that SAM is the first substrate and DOIA is the second. The BtrN reaction with [3-2H]DOIA generated nonlabeled, monodeuterated and dideuterated 5‘-deoxyadenosines, while no deuterium was incorporated by incubation of nonlabeled DOIA in the deuterium oxide buffer. These results indicated that the hydrogen atom at C-3 of DOIA was directly transferred to 5‘-deoxyadenosine to give the radical intermediate of DOIA. Generation of nonlabeled and dideuterated 5‘-deoxyadenosines proved the reversibility of the hydrogen abstraction step. The present study suggests that BtrN is an unusual radical SAM dehydrogenase catalyzing the oxidation of the hydroxyl group by a radical mechanism. This is the first report of the mechanistic study on the oxidation of a hydroxyl group by a radical SAM enzyme.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja072481t