Butirosin A

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.

Using a comparative genetics approach, one or more of the BtrA, BtrL, BtrP, and BtrV proteins encoded in the butirosin biosynthetic gene cluster (btr) from Bacillus circulans SANK72073 were identified as being responsible for an O-ribosylation process leading to the formation of ribostamycin, a key intermediate in this, and related antibiotic biosynthetic pathways. Functional analysis of the recombinantly expressed proteins revealed that both BtrL and BtrP were responsible for the ribosylation of neamine, using 5-phosphoribosyl-1-diphosphate (PRPP) as the ribosyl donor. Further detailed analysis indicated that this process occurs via two discrete steps: with BtrL first catalyzing the phosphoribosylaion of neamine to form 5''-phosphoribostamycin, followed by a BtrP-catalyzed dephosphorylation to generate ribostamycin. To the best of our knowledge, this is the first time that the functional characterization of a glycosyltransferase from an aminoglycoside biosynthetic gene cluster has been reported.

The HIV-1 Dimerization Initiation Site (DIS) is an intriguing, yet underutilized, viral RNA target for potential antiretroviral therapy. To study the recognition features of this target and to provide a quantitative, rapid, and real-time tool for the discovery of new binders, a fluorescence-based assay has been constructed. It relies on strategic incorporation of 2-aminopurine, an isosteric fluorescent adenosine analogue, into short hairpin RNA constructs. These oligomers self-associate to form a kissing loop that thermally rearranges into a more stable extended duplex, thereby mimicking the association and structural features of the native RNA sequence. We demonstrate the ability of two fluorescent DIS constructs, DIS272(2AP) and DIS273(2AP), to report the binding of known DIS binders via changes in their emission intensity. Binding of aminoglycosides such as paromomycin to DIS272(2AP) results in significant fluorescence enhancement, while ligand binding to DIS273(2AP) results in fluorescence quenching.