Glycosylation Steps during Spiramycin Biosynthesis in Streptomyces ambofaciens : Involvement of Three Glycosyltransferases and Their Interplay with Two Auxiliary Proteins

Abstract

Streptomyces ambofaciens synthesizes spiramycin, a 16-membered macrolide antibiotic used in human medicine. The spiramycin molecule consists of a polyketide lactone ring (platenolide) synthesized by a type I polyketide synthase, to which three deoxyhexoses (mycaminose, forosamine, and mycarose) are attached successively in this order. These sugars are essential to the antibacterial activity of spiramycin. We previously identified four genes in the spiramycin biosynthetic gene cluster predicted to encode glycosyltransferases. We individually deleted each of these four genes and showed that three of them were required for spiramycin biosynthesis. The role of each of the three glycosyltransferases in spiramycin biosynthesis was determined by identifying the biosynthetic intermediates accumulated by the corresponding mutant strains. This led to the identification of the glycosyltransferase responsible for the attachment of each of the three sugars. Moreover, two genes encoding putative glycosyltransferase auxiliary proteins were also identified in the spiramycin biosynthetic gene cluster. When these two genes were deleted, one of them was found to be dispensable for spiramycin biosynthesis. However, analysis of the biosynthetic intermediates accumulated by mutant strains devoid of each of the auxiliary proteins (or of both of them), together with complementation experiments, revealed the interplay of glycosyltransferases with the auxiliary proteins. One of the auxiliary proteins interacted efficiently with the two glycosyltransferases transferring mycaminose and forosamine while the other auxiliary protein interacted only with the mycaminosyltransferase.Macrolide antibiotics consist of a polyketide macrolactone ring to which one or more deoxysugars are attached. They inhibit protein synthesis by binding to the large subunit of the ribosome and can also, in some cases, interfere with the assembly of the large subunit (reviewed in reference19). As with many other natural products, glycosylation of macrolides is essential for their biological activity. The determination of the three-dimensional structure of the 50S ribosomal unit in complex with various macrolides showed that the sugar moieties are involved in specific interactions with the 23S rRNA (11, 38). All macrolides block the ribosomal peptide exit tunnel, thus preventing peptide chain elongation. In addition to their role in binding, the sugars are also important for steric hindrance in the tunnel. For instance, it was shown that the C-5 disaccharide moieties of some 16-membered macrolides, such as spiramycin or tylosin, extend in the tunnel toward the peptidyl transferase center, and a correlation could be established between the length of the macrolide saccharide side chain and the number of amino acids which could be incorporated before peptide chain elongation was blocked (11).One of the most common resistance mechanisms to macrolide antibiotics encountered in pathogenic bacteria involves target modification. These modifications could include mutation or m...

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

“…Similarly, other glycosyltransferases have been found to require another auxiliary protein for their activity (39)(40)(41). A recent study showed the role of two auxiliary proteins, Srm6 and Srm28, in the activation of two glycosyltransferases, Srm5 and Srm29, respectively, and both are required for the biosynthesis of spiramycin by Streptomyces ambofaciens (42). Activity of the glycosyltransferase EryCIII, which is required for biosynthesis of the antibiotic erythromycin D, is dependent upon an auxiliary protein, EryCII (43).…”

“…Activity of the glycosyltransferase EryCIII, which is required for biosynthesis of the antibiotic erythromycin D, is dependent upon an auxiliary protein, EryCII (43). Although the roles of these auxiliary proteins are not clear, it has been proposed that these auxiliary proteins are required to induce conformational changes of the glycosyltransferases, which consequently activate these enzymes (36,42).…”

Regulation of the Biosynthesis of the Macrolide Antibiotic Spiramycin in Streptomyces ambofaciens

“…Spiramycins I, II, and III were used as standards for quantification by HPLC. The nomenclature used for the genes of the spiramycin biosynthetic gene cluster is different from the one published earlier (25) but is the one used by Nguyen et al (32).…”

The adnAB Locus, Encoding a Putative Helicase-Nuclease Activity, Is Essential in Streptomyces

“…The two PCR fragments were cloned into the pGEM-T Easy vector and then released by HindIII/EcoRV or EcoRV/NheI digestions. The excisable cassette att3-aac conferring apramycin resistance was excised from pOSV234 (29) by EcoRV digestion. The above-mentioned three fragments were simultaneously ligated with the HindIII/NheI-digested vector pWED2 (30), which confers puromycin resistance to generate an adnAB deletion vector.…”