Characterization and mechanistic study of a radical SAM dehydrogenase in the biosynthesis of butirosin.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Yokoyama, K; Numakura, M; Kudo, F; Ohmori, D; Eguchi, T

Published Date

  • December 12, 2007

Published In

Volume / Issue

  • 129 / 49

Start / End Page

  • 15147 - 15155

PubMed ID

  • 18001019

Electronic International Standard Serial Number (EISSN)

  • 1520-5126

Digital Object Identifier (DOI)

  • 10.1021/ja072481t


  • eng

Conference Location

  • United States