Published on Web 11/15/2007
Characterization and Mechanistic Study of a Radical SAM
Dehydrogenase in the Biosynthesis of Butirosin
Kenichi Yokoyama,† Mario Numakura,† Fumitaka Kudo,† Daijiro Ohmori,§ and
Tadashi Eguchi*,‡
Contribution from the Department of Chemistry, and Department of Chemistry & Materials
Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo152-8551, Japan, and
Department of Chemistry, Juntendo UniVersity, Inba, Chiba 270-1695, Japan
Received April 9, 2007; E-mail: eguchi@cms.titech.ac.jp
Abstract: 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.
Introduction
led to the identification of several other DOS-containing
aminoglycoside gene clusters.8-12 Comparative genetics of the
2-Deoxystreptamine (DOS) containing aminoglycosides con-
stitute the largest subgroup of aminoglycoside antibiotics. This
group of aminoglycosides includes kanamycin, neomycin, and
gentamicin, which are clinically important antibiotic agents used
against bacterial and some protozoal infections.1 In addition to
these classical roles they are also attracting attention for their
RNA and DNA recognition ability, which can be applied for
anti-plasmids2,3 and anti-HIV agents.4,5 Increased attention to
the biological activity of aminoglycosides makes their biosyn-
thetic machinery more attractive for creating structurally diverse
substances by engineered biosynthesis.
open reading frames (ORFs) in those gene clusters, combined
with gene disruption experiments and in vitro assays of
overexpressed enzymes, led to reasonable assignments of each
ORF in the butirosin biosynthetic gene cluster to each biosyn-
thetic reaction.8,13 However, the functions of several ORFs still
remain uncharacterized.
BtrN, a functionally unknown enzyme encoded in the
butirosin biosynthetic gene cluster, has an amino acid sequence
motif (CXXXCXXC), which is typical of the radical S-adenosyl
methionine (SAM) superfamily.14 The radical SAM superfamily
is an emerging group of enzymes that catalyze a wide range of
The biosynthetic gene cluster of butirosin was the first of
the DOS-containing aminoglycosides to be identified,6,7 which
(6) Ota, Y.; Tamegai, H.; Kudo, F.; Kuriki, H.; Koike-Takeshita, A.; Eguchi,
T.; Kakinuma, K. J. Antibiot. 2000, 53, 1158-1167.
† Department of Chemistry, Tokyo Institute of Technology.
§ Juntendo University.
(7) Tamegai, H.; Nango, E.; Kuwahara, M.; Yamamoto, H.; Ota, Y.; Kuriki,
H.; Eguchi, T.; Kakinuma, K. J. Antibiot. 2002, 55, 707-714.
(8) Huang, F.; Haydock, S. F.; Mironenko, T.; Spiteller, D.; Li, Y.; Spencer,
J. B. Org. Biomol. Chem. 2005, 3, 1410-1418.
‡ Department of Chemistry & Materials Science, Tokyo Institute of
Technology.
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