Identification of the Function of Gene lndM2 Encoding a Bifunctional Oxygenase-Reductase Involved in the Biosynthesis of the Antitumor Antibiotic Landomycin E by Streptomyces globisporus 1912 Supports the Originally Assigned Structure for Landomycinone

Abstract

The angucycline antibiotic family of the landomycins displays potent antitumor activity. To elucidate early post polyketide synthase (PKS) tailoring steps of the landomycin E biosynthetic pathway in Streptomyces globisporus 1912, the mutant S. globisporus M12 was prepared through gene replacement experiment of lndM2. It encodes an enzyme with putative oxygenase and reductase domains, according to sequencing of the gene and its counterpart lanM2 from S. cyanogenus S136 landomycin A biosynthetic gene cluster. The isolation of the novel shunt products 11-hydroxytetrangomycin and 4-hydroxytetrangomycin along with the well-known angucyclines tetrangomycin and tetrangulol from the culture of S. globisporus M12 provides evidence for the © 2005 American Chemical Society jrohr2@uky.edu. Supporting Information Available: Original NMR spectra obtained after the acetate feeding experiments on landomycin A and a newly generated 1 H NMR spectrum of natural landomycinone supporting the original landomycin structure. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript involvement of lndM2 in the early biosynthetic pathway of the landomycins, in particular in the formation of the alicyclic 6-hydroxy function of the landomycin aglycon. We therefore propose LndM2 to be responsible for both hydroxylation of the 6-position and its subsequent reduction. These reactions are necessary before the glycosylation reactions can occur. The results are in agreement with the originally published structure of landomycin but do not support the recently suggested revised structure.

On the Acceptor Substrate of C‐Glycosyltransferase UrdGT2: Three Prejadomycin C‐Glycosides from an Engineered Mutant of Streptomyces globisporus 1912 ΔlndE(urdGT2)

“…[5] In particular, it was recently shown that the non-glycosylated angucyclinones tetrangomycin (11) and rabelomycin (12) can be further converted into landomycin E (1) by the lndF mutant F133, and thus 11 and 12 are biosynthetic intermediates. [12] The results described here confirm this description of a-compared to the urdamycin pathway-later first glycosylation step. The fact that none of the new prejadomycin C-glycosides including monoglycoside 7 could be further modified by the oxygenases that exist in S. globisporus ΔlndE (urdGT2), for example, by LndM2 and LndZ4/Z5, shows that these oxygenases require non-glycosylated substrates.…”

“…Major structural differences were found in the assembly of the sugar moieties and in the oxygenation pattern of the polyketide core moiety. [1,4,5,[10][11][12] The availability of the biosynthetic gene clusters of urdamycins and landomycins [7,[13][14][15][16][17] made it possible to track down the genes encoding the biosynthetic enzymes responsible for the unique structural features of the two types of compounds, which are also responsible for the significant variations in the biological activities of these related anti-cancer drugs. [18] The overall structural and biosynthetic similarity between these two closely related sets of antibiotics allowed several successful combinatorial, biosynthetic gene-combination experiments, leading to new hybrid molecules.…”

“…Regarding the oxygenases of the landomycin biosynthesis, it was found that oxygenase Lan/ LndZ5 is responsible for the 11-hydroxylation, [5] and Lan/LndM2 for the 6-hydroxylation, [12] while Lan/LndE most likely catalyzes the 12-oxygenation (quinone formation), which was assumed to be the first oxygenation step in the landomycin biosynthesis. All oxygenations steps except the 11-hydroxylation occur before Lan/LndGT2 attaches the first sugar moiety.…”

“…[8,19] To find out whether prejadomycin (6) is an early intermediate of the landomycin pathway or an early intermediate of a shunt pathway branching from the landomycin pathway (Scheme 1), we performed a crossfeeding experiment, in which prejadomycin (6, obtained from Streptomyces globisporus ΔlndE) was fed to an early block mutant of the landomycin biosynthetic pathway (the lndF mutant F133). [12] In this mutant the PKS-associated fourth ring cyclase gene lndF was inactivated, and consequently it cannot produce any useful polyketides. But the mutant expresses all downstream enzymes necessary for the completion of landomycin E biosynthesis when it is fed with an intermediate containing a completely cyclized polyketide intermediate.…”

Premithramycinone G, an Early Shunt Product of the Mithramycin Biosynthetic Pathway Accumulated upon Inactivation of Oxygenase MtmOII

“…For the instruments/NMR methods used, see Ref. [42]. The labeled sodium [1,2-13 C 2 ]acetate used in the incorporation experiment was obtained from Isotec (Miamisburg, OH, USA).…”