Pseudoglycosyltransferase Catalyzes Nonglycosidic C–N Coupling in Validamycin A Biosynthesis

Shumpei Asamizu, Jongtae Yang, Khaled H. Almabruk, Taifo Mahmud
2011 Journal of the American Chemical Society  
Glycosyltransferases are ubiquitous in nature. They catalyze a glycosidic bond formation between sugar donors and sugar or non-sugar acceptors to produce oligo/polysaccharides, glycoproteins, glycolipids, glycosylated natural products, and other sugar-containing entities. However, a trehalose 6-phosphate synthase-like protein has been found to catalyze an unprecedented nonglycosidic C-N bond formation in the biosynthesis of the aminocyclitol antibiotic validamycin A. This dedicated
more » ... yltransferase' catalyzes a condensation between GDP-valienol and validamine 7-phosphate to give validoxylamine A 7′-phosphate with net retention of the 'anomeric' configuration of the donor cyclitol in the product. The enzyme operates in sequence with a phosphatase, which dephosphorylates validoxylamine A 7′-phosphate to validoxylamine A. VldB, VldE, and VldH production and purification-pET20-vldB, pRSETB-vldE and pRSETB-vldH were used to transform E. coli BL21(DE3) pLysS. Transformants were grown overnight at 37 °C on LB agar plate containing 100 μg/mL ampicillin and 25 μg/mL chloramphenicol. A single colony was inoculated into 3 mL of LB medium and cultured at 37 °C for 6 h and then 1 mL of seed culture was transferred into 100 mL of LB medium in a 500 mL flask and grown at 28 °C until OD 600 reached 0.6. Then, the temperature was reduced to 18 °C and after 2 h adaptation 0.1 mM IPTG was added to induce the C-terminal hexahistidine-tagged VldB and the N-terminal hexahistidine-tagged VldE and VldH proteins. After further growth for 14 h, the cells were harvested by centrifugation (5,000 rpm, 10 min, 4 °C) and stored at −80 °C until used. Cell pellets from 50 mL of culture were washed with 1 mL of binding (B) buffer (40 mM HEPES (pH 8.0), 300 mM NaCl, 10% glycerol, and 10 mM imidazole) and centrifuged (6,000 rpm, 3 min, 4 °C). Then, 1 mL of B buffer was added and the mixtures were sonicated (8 watts, 15 sec, 4 times). After centrifugation (14,500 rpm, 20 min, 4 °C), 0.8 mL of the supernatants were each mixed with 0.2 mL of B buffer-equilibrated Ni-NTA resin (QIAGEN) and incubated for 1 h at 4 °C. After incubation, the mixtures were centrifuged (4,000 rpm, 3 min, 4 °C) and the supernatants were discarded. One mL of washing (W) buffer (40 mM HEPES pH 8.0, 300 mM NaCl, 10% glycerol, and 20 mM imidazole in the case of VldE and VldH or 100 mM imidazole in the case of VldB) was added and the mixture centrifuged (4,000 rpm, 3 min, 4°C ). This washing step was repeated three times. Subsequently, 0.5 mL of elution (E) buffer (40 mM HEPES pH 8.0, containing 300 mM NaCl, 10% glycerol, and 500 mM imidazole) was added to elute the desired proteins. This elution step was repeated twice. Eluted VldB, VldE, and VldH were dialyzed against 1 L of dialysis buffer (10 mM Tris-HCl pH 7.5, 0.1 mM DTT, 1 mM MgCl 2 ) 3 times for 3 h each. Purified proteins were analyzed by SDS-PAGE and concentrated by ultrafiltration using Amicon YM-10 (Millipore). Protein concentration was determined using the Bradford assay (BIO-RAD) with BSA as standard.
doi:10.1021/ja203574u pmid:21766819 pmcid:PMC3162038 fatcat:iuhapwp22bditd43vgeqf6ekq4