Biosynthesis of albomycin δ(2) provides a template for assembling siderophore and aminoacyl-tRNA synthetase inhibitor conjugates

Abstract

"Trojan horse" antibiotic albomycins are peptidyl nucleosides consisting of a highly modified 4'-thiofuranosyl cytosine moiety and a ferrichrome siderophore that are linked by a peptide bond via a serine residue. While the latter component serves to sequester iron from the environment, the seryl nucleoside portion is a potent inhibitor of bacterial seryl-tRNA synthetases, resulting in broad-spectrum antimicrobial activities of albomycin δ(2). The isolation of albomycins has revealed this biological activity is optimized only following two unusual cytosine modifications, N4-carbamoylation and N3-methylation. We identified a genetic locus (named abm) for albomycin production in Streptomyces sp. ATCC 700974. Gene deletion and complementation experiments along with bioinformatic analysis suggested 18 genes are responsible for albomycin biosynthesis and resistance, allowing us to propose a potential biosynthetic pathway for installing the novel chemical features. The gene abmI, encoding a putative methyltransferase, was functionally assigned in vitro and shown to modify the N3 of a variety of cytosine-containing nucleosides and antibiotics such as blasticidin S. Furthermore, a ΔabmI mutant was shown to produce the descarbamoyl-desmethyl albomycin analogue, supporting that the N3-methylation occurs before the N4-carbamoylation in the biosynthesis of albomycin δ(2). The combined genetic information was utilized to identify an abm-related locus (named ctj) from the draft genome of Streptomyces sp. C. Cross-complementation experiments and in vitro studies with CtjF, the AbmI homologue, suggest the production of a similar 4'-thiofuranosyl cytosine in this organism. In total, the genetic and biochemical data provide a biosynthetic template for assembling siderophore-inhibitor conjugates and modifying the albomycin scaffold to generate new derivatives.

Figure 1

Structure of albomycins. The dashed line indicates the site of cleavage by bacterial peptidases to give a ferrichrome-type hydroxamate siderophore and the seryl thionucleoside compound SB-217452. The R in SB-217452 is identical to albomycin δ₂.

Figure 2

Biosynthetic gene clusters of albomycins and a putative albomycin-type analog. (a) Genetic organization of the albomycin (abm) gene cluster in Streptomyces sp. ATCC 700974 and (b) ctj gene cluster in Streptomyces sp. strain C. The homologous abm and ctj genes are filled with same colors. Genes unique to each cluster are filled in grey.

Figure 2

Biosynthetic gene clusters of albomycins and a putative albomycin-type analog. (a) Genetic organization of the albomycin (abm) gene cluster in Streptomyces sp. ATCC 700974 and (b) ctj gene cluster in Streptomyces sp. strain C. The homologous abm and ctj genes are filled with same colors. Genes unique to each cluster are filled in grey.

Figure 3

HPLC analysis of extracts of the ΔabmE mutant strain. The ΔabmE mutant strain was complemented with either abmE or homologous gene ctjE and shown to restore the albomycin δ₂ production in contrast to the empty vector pSE34.

Figure 4

ESI-MS analysis of a partially purified fraction from ΔabmI mutant that produces the descarbamoyl-desmethyl-albomycin compound. The insert is the simulated isotopic distribution of the MS ions according to the expected molecular formula. See Supporting Figure S6 for HPLC profile of the active fraction.

Figure 5

HPLC analysis of AbmI in vitro reaction with cytidine as a substrate. I. Cytidine standard (C); II. N3-methylacytidine (³mC); III. AbmI reaction supplied with SAH nucleosidase after 4-hour incubation; IV. Reaction conditions are identical to III except that AbmI was heat-inactivated prior to the incubation. A major peak adjacent to cytidine in III and IV is adenine due to SAH degradation. Other minor peaks in III are also result of the SAH nucleosidase.

Figure 6

AbmI specificity toward cytosine-containing compounds in in vitro reaction. (a) Structures of the cytosine-containing nucleosides. The red ball denotes the site of methylation. LCMS data are provided in Supporting Figure S11. (b) Positive ESI-MS analysis of the deuterium labeled methyl blasticidin S product of an AbmI in vitro reaction. Substrates d₃-SAM and ¹H₃-SAM were pre-mixed in a 2:3 ratio. The HPLC profile of the reaction is provided in Supporting Figure S14.

Figure 7

The proposed albomycin biosynthetic pathway in Streptomyces sp. ATCC 700974. Three hypothetical pathways leading to the formation of the C7N amino acid are presented in Supporting Figure S15.