Characterization of a Sugar‐O‐methyltransferase TiaS5 Affords New Tiacumicin Analogues with Improved Antibacterial Properties and Reveals Substrate Promiscuity

Abstract

The 18-membered macrocyclic glycoside tiacumicin B, an RNA polymerase inhibitor, is of great therapeutic significance in treating Clostridium difficile infections. The recent characterization of the tiacumicin B biosynthetic gene cluster from Dactylosporangium aurantiacum subsp. hamdenensis NRRL 18085 revealed the functions of two glycosyltransferases, a C-methyltransferase, an acyltransferase, two cytochrome P450s, and a tailoring dihalogenase in tiacumicin biosynthesis. Here we report the genetic confirmation and biochemical characterization of TiaS5 as a sugar-O-methyltransferase, requisite for tiacumicin B biosynthesis. The tiaS5-inactivation mutant is capable of producing 14 tiacumicin analogues (11 of which are new), all lacking the 2'-O-methyl group on the internal rhamnose moiety. Notably, two tiacumicin analogues exhibit improved antibacterial properties. We have also biochemically verified TiaS5 as an S-adenosyl-L-methionine-dependent O-methyltransferase, requiring divalent metal ions for activity. Substrate probing revealed TiaS5 to be a promiscuous enzyme, recognizing 12 tiacumicin analogues. These findings unequivocally establish that TiaS5 functions as a 2'-O-methyltransferase and provide direct biochemical evidence that TiaS5-catalyzed methylation is a tailoring step after glycosyl coupling in tiacumicin B biosynthesis.

Assisted Reproduction: What factors interfere in the professional's decisions? Are single women an issue?

“…On the other hand, the fact that Brazilians (60%) showed more tolerance could lead to the assumption that the culture in which they live influences with the decisions they make. The widespread occurrence of Brazilian families "headed by women" [14] and the strong association existing between reproduction and woman in Brazil [15] cause the single-woman-with-no-partner situation not to be perceived as unusual, as might be the case in some European countries.…”

“…For such techniques to be conceded there must be a medical indication therefor and voluntary and informed consent is mandatory for both beneficiaries when the woman receiving treatment is married or in a domestic partnership [15,16]. …”

“…However, it has been observed that individual realization is also being sought by means of this technology, a phenomenon which has lead professionals to express caution about the procedure in such cases. The impossibility of procreating is indeed an obstacle for a woman's plans, and the recourse to assisted reproduction can be commonly observed within cultural contexts marked by individualism and autonomy [15]. Moreover, the incidence of births to single or unmarried persons has grown worldwide, including among never-married, college-educated, professional women.…”

“…However, one must bear in mind that most of these professionals work in private centers, which means they cater for a higher income population. Thus the question remains whether this autonomy and freedom would be equally valued in the case of a woman in a poor setting [15]. …”

Totalsynthese des Aglykons von Tiacumicin B (Lipiarmycin A3/ Fidaxomicin)

“…Diese Arbeiten sollen eine Grundlage für nachfolgende Struktur-Aktivitäts(SAR)-Studien schaffen, die wiederum zur Entdeckung von Tiacumicin-B-Derivaten mit verbesserten biopharmazeutischen Eigenschaften führen kçnnten. [14] Gleichzeitig mit uns haben auch die Forschungsgruppen um Gademann und Zhu in unabhängigen Studien Synthesewege zu 2 bzw. dessen 18S-Isomer erarbeitet.…”