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.

The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (K(m) of 338microM and V(max) of 601micromol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.

BtrN is a radical SAM ( S-adenosyl- l-methionine) enzyme that catalyzes the oxidation of 2-deoxy- scyllo-inosamine (DOIA) into 3-amino-2,3-dideoxy- scyllo-inosose (amino-DOI) during the biosynthesis of 2-deoxystreptamine (DOS) in the butirosin producer Bacillus circulans. Recently, we have shown that BtrN catalyzes the transfer of a hydrogen atom at C-3 of DOIA to 5'-deoxyadenosine, and thus, the reaction was proposed to proceed through the hydrogen atom abstraction by the 5'-deoxyadenosyl radical. In this work, the BtrN reaction was analyzed by EPR spectroscopy. A sharp double triplet EPR signal was observed when the EPR spectrum of the enzyme reaction mixture was recorded at 50 K. The spin coupling with protons partially disappeared by reaction with [2,2- (2)H 2]DOIA, which unambiguously proved the observed signal to be a radical on C-3 of DOIA. On the other hand, the EPR spectrum of the [4Fe-4S] cluster of BtrN during the reaction showed a complex signal due to the presence of several species. Comparison of signals derived from a [4Fe-4S] center of BtrN incubated with various combinations of products (5'-deoxyadenosine, l-methionine, and amino-DOI) and substrates (SAM and DOIA) indicated that the EPR signals observed during the reaction were derived from free BtrN, a BtrN-SAM complex, and a BtrN-SAM-DOIA complex. Significant changes in the EPR signals upon binding of SAM and DOIA suggest the close interaction of both substrates with the [4Fe-4S] cluster.