GoKawiilIn vitro GT glycosylation assays
For GT assays with TDP glucose; GT enzyme (5–20 µM), TDP-glucose (2 mM), MgCl 2 (1 mM) and polyene substrates, including amphotericin B (AmB), AmB-aglycone, nystatin A1 (NysA1) and mycoheptin and dihydromycoheptin, at 100 µM concentration were incubated in a total reaction volume of 50 µl in Tris-HCl buffer (50 mM, pH 7.4) at 30 °C for 24 h. For TDP sugars other than TDP glucose with available sugar-1-phosphates, an extra step was required in which the TDP sugar was produced from the corresponding sugar-1-phosphate. In this first step, deoxythymidine triphosphate (2 mM), sugar-1-phosphate (2 mM), MgCl 2 (2.2 mM) and Cps2L (50 µM) in a total volume of 50 µl in Tris-HCl buffer (50 mM Tris-HCl, pH 7.4) were incubated for 24 h at 37 °C in a shaking thermomixer. After 24 h, the temperature was adjusted to 30 °C and GT (5–20 µM) and polyene (100 µM) were added. Assays were then incubated for a further 24 h at 30 °C. For TDP sugars with no available sugar-1-phosphate, TDP sugars were produced from the corresponding sugar. In this modified first step, deoxythymidine triphosphate (2 mM), ATP (2 mM), sugar (2 mM), MgCl 2 (2.2 mM), NahK_ATCC15697 or GalkSpe4 (50 µM) and Cps2L (50 µM) in a total volume of 50 µl in Tris-HCl buffer (50 mM Tris-HCl, pH 7.4) was incubated for 24 h at 37 °C in a shaking thermomixer. After 24 h, the temperature was adjusted to 30 °C and GT (5–20 µM) and polyene (100 µM) were added. Assays were then incubated for a further 24 h at 30 °C.
For all assays (1, 2 or 3 steps), reactions were quenched by heating to 95 °C for 5 min, followed by the addition of 1 reaction volume of methanol. The protein was then pelleted by centrifugation, and the supernatant was analysed by analytical RP-HPLC and LC–HRMS. Assays were analysed on a Shimadzu Analytical ultrahigh-performance liquid chromatography (UHPLC) apparatus with a Kinetex 5 μm XB-C18 100 × 4.6 mm (Phenomenex) column, with a flow rate of 1 ml min−1, using a solvent system of water and methanol with 0.1% formic acid and a gradient of 60–78% methanol over 12 min. Heptaenes were monitored at 405 nm and pentaenes at 350 nm. LC–HRMS was performed on an Agilent 1290 Infinity II coupled to a 6560 Ion Mobility quadrupole time of flight (Q-TOF) LC–MS apparatus and 1290 Infinity II HPLC coupled to a 6546 LC–Q-TOF (Agilent Instruments), using a Luna Omega 5 μm 100 × 2.1 mm (Phenomenex) column, with a flow rate of 0.5 ml min−1, using a solvent system of water and methanol and a gradient of 60–95% methanol over 15 min. On LC chromatograms, tetraenes were monitored at 304 nm, pentaenes at 350 nm and heptaenes at 405 nm.
GT-catalysed reverse glycosylation of polyenes
Each of the polyenes (25–100 μM, kasufungin B, NysA3, semipurified selvamicin) were incubated with TDP (2 mM), MgCl 2 (1 mM), GT (KfuSV, NysSV, SelSV, 25 μM) in 50 mM Tris-HCl buffer pH 7.5–8, 30 °C overnight, in a 50 μl reaction. The reactions were quenched with methanol (50 μl) and centrifuged (10,000 rpm, 5 min). The supernatant was analysed by HPLC and masses were confirmed by LC–MS using the same conditions as mentioned above in the in vitro GT glycosylation assays section.
MycS3 assay with dihydromycoheptin and TDP- l -digitoxose
Reverse glycosylation of NysA3 was performed to generate TDP-l-digitoxose. A 200–300 μM solution of NysA3 was incubated with TDP (2 mM), MgCl 2 (1 mM) and KfuSV (50 μM) in 50 mM Tris-HCl buffer pH 7.5–8, at 30 °C overnight, in a 2-ml reaction. The reactions were quenched with an equal volume of methanol to inactivate enzyme and centrifuged (10,000 rpm, 5 min). The supernatant was concentrated under vacuum to reduce the volume to one-quarter, applied to silica filled in a small syringe (silica C18 100 Å, 30 μm, silica bed 4 ml) and washed twice with an equal volume of H 2 O to elute TDP-l-digitoxose. The filtrate volume was reduced to one-fifth under vacuum and used for MycS3 assays.
For assays with MycS3, a solution of dihydromycoheptin (50 µM) in MycS3 reaction buffer (1 mM MgCl 2 , 50 mM Tris-HCl, pH 7.5) was incubated with the TDP-l-digitoxose containing concentrated filtrate (25 μl) and MycS3 (25 μM) at 30 °C overnight in 50-μl reactions. The reactions were quenched with methanol (50 μl) and centrifuged (10,000 rpm, 5 min) before the supernatant was analysed by HPLC and LC–MS. In separate experiments, dried S. netropsis (DSM40846) methanolic extract containing crude dihydromycoheptin, was resuspended in MycS3 reaction buffer (1 mM MgCl 2 , 50 mM Tris-HCl, pH 7.5) was incubated with the TDP-l-digitoxose containing concentrated filtrate (25 μl) and MycS3 (25 μM) at 30 °C overnight in 50-μl reactions. The reactions were quenched with methanol (50 μl) and centrifuged (10,000 rpm, 5 min) before the supernatant was analysed by LC–HRMS. HPLC analysis was performed on a Shimadzu Analytical UHPLC with a Kinetex 5 μm XB-C18 100 × 4.6 mm (Phenomenex) column, with a flow rate of 1 ml min−1, using a solvent system of water and methanol (+0.1% formic acid) and a gradient of 60–78% methanol over 12 min, with UV monitored at 350 nm. LC–HRMS was performed on an Agilent 1290 Infinity II coupled to a 6560 Ion Mobility Q-TOF LC–MS and 1290 Infinity II HPLC coupled to a 6546 LC/Q-TOF (Agilent Instruments), using a Luna Omega 5 μm 100–2.1 mm (Phenomenex) column, with a flow rate of 0.5 ml min−1, using a solvent system of water and methanol and a gradient of 60–95% methanol over 15 min.
In vitro PcsA reaction conditions
Enzyme activity assays were carried out in a reaction containing 100 µM polyene (pimaricin, rimocidin, CE-108, NysA1, NysA3, mycoheptin, dihydromycoheptin, kasufungin B or candicidin) and l-glutamine, l-glutamic acid γ-hydroxamate or l-glutamic acid γ-hydrazide (2 mM), ATP (4 mM), MgCl 2 (10 mM) and 25 µl of eluted protein in Tris buffer (125 mM Tris and 25 mM NaCl, pH 7) in a total volume of 50 µl. In addition, l-glutamine analogues (l-glutamic acid γ-methylamide, l-glutamic acid γ-ethylamide (l-theanine), l-glutamic acid γ-methyl ester and l-glutamic acid γ-ethyl ester) were also tested but no activity was observed. The reactions were quenched with methanol (50 µl) after overnight incubation at 30 °C, and centrifuged (10,000 rpm for 5 min). Assays were analysed on a Shimadzu Analytical UHPLC with a Kinetex 5 μm XB-C18 100 × 4.6 mm (Phenomenex) column, using 1 ml min−1 flow rate with a solvent system of water and methanol each containing 0.1% formic acid, using a gradient of 60–78% methanol over 12 min. LC–HRMS was performed on an Agilent 1290 Infinity II coupled to a 6560 Ion Mobility Q-TOF LC–MS instrument, using a Luna Omega 5 μm 100 × 2.1 mm (Phenomenex) column, using a 0.5 ml min−1 flow rate with a solvent system of water and methanol each containing 0.1% formic acid, and a gradient of 60–80% methanol over 15 min.
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