The in vitro characterization of polyene glycosyltransferases AmphDI and NysDI

Abstract

The overproduction, purification, and in vitro characterization of the polyene glycosyltransferases (GTs) AmphDI and NysDI are reported. A novel nucleotidyltransferase mutant (RmlA Q83D) for the chemoenzymatic synthesis of unnatural GDP-sugar donors in conjunction with polyene GT-catalyzed sugar exchange/reverse reactions allowed the donor and acceptor specificities of these novel enzymes to be probed. The evaluation of polyene GT aglycon and GDP-sugar donor specificity revealed some tolerance to aglycon structural diversity, but stringent sugar specificity, and culminated in new polyene analogues in which L-gulose or D-mannose replace the native sugar D-mycosamine.

Figure 1. Polyene GT-catalyzed reverse reactions

(A) Schematic of polyene GT-catalyzed conversion of 1, 2 or 4 to deglycosylated products 7, 8 or 9, respectively; (B) Schematic of polyene GT-catalyzed conversion of candicidin complex (3-I, 3-II, 3 and 3-IV) to deglycosylated complex (12-I, 12-II, 12 and 12-IV); (C) HPLC analyses of AmphDI-T2 NDP-specificity in GT-catalyzed reverse reactions. In this example, 20 μM AmB (1) was incubated with 5 μM of AmphDI-T2 without NDP (i) or in the presence of 1 mM of ADP (ii), CDP (iii), UDP (iv), TDP (v), GDP (vi) or dGDP (vii), at 30 °C overnight; (D) HPLC analyses of polyene GT-catalyzed reverse reactions with AmB (1) and different polyene GTs. For this study, 20 μM AmB (1) was incubated with 1 mM of GDP in the presence of 5 μM AmphDI (i), AmphDI-T2 (ii), NysDI (iii), NysDI-T2 (iv) or without GT (v), at 30 °C overnight; (E) HPLC analyses of AmphDI-T2 aglycon specificity in GT-catalyzed reverse reactions. In this study, 20 μM nystatin (2), 50 μM pimaricin (4) or 20 μM of candicidin complex (3-I, 3-II, 3 and 3-IV) were incubated with 1 mM GDP in the absence or presence of 5 μM AmphDI-T2: (i) 2, no enzyme (control), (ii) 2, AmphDI-T2, (iii) 4, no enzyme (control), (iv) 4, AmphDI-T2, (v) candicidin complex (3-I, 3-II, 3 and 3-IV), no enzyme (control), (iii) candicidin complex (3-I, 3-II, 3 and 3-IV), AmphDI-T2.

Figure 2. Polyene GT-catalyzed Sugar Exchange Reactions

(A) Schematic of polyene GT-catalyzed sugar exchange reaction. (B) HPLC analyses of AmphDI-T2 catalyzed sugar exchange reactions. In this study, 20 μM AmB (1) was co-incubated with 5 μM of AmphDI-T2 in the presence of 1 mM of (i) GDP-α-D-altrose (18), (ii) GDP-α-D-talose (19), (iii) GDP-β-L-gulose (17), (iv) GDP-β-L-mannose (20), (v) GDP-α-D-glucose (21), (vi) GDP-β-L-glucose (23), (vii) GDP-α-D-mannose (22).

Scheme 1. Naturally-occurring polyene macrolides

Amphotericin B (1), nystatin A1 (2), candicidin/FR-008 (3), pimaricin (4), rimocidin (5) and filipin III (6) are all produced by Streptomyces strains and genetic loci for 1–5 have been characterized. The corresponding mycosaminyltransferases (AmphDI, NysDI, FscMI, PimK and RimE, respectively) responsible for glycoside formation are highlighted.

Scheme 2. Chemical and chemoenzymatic preparation of GDP-sugars

(A) The chemical synthesis of GDP-L-β-gulose (17). (i) Ac₂O/pyridine; HBr/AcOH; HPO₂(OBn)₂, CF₃SO₃Ag, Me₃C₅H₂N/CH₂Cl₂; (ii) H₂/Pd-C; AG 50W-X8 (Et₃NH⁺); (iii) GDP-morpholidate (16) and 1H-tetrazole/pyridine. (B) The chemoenzymatic synthesis of GDP-sugars. Generally, 6 mM of chemically synthesized sugar-1 phosphate was incubated with 8 mM of GTP in the presence of 20 μM RmlA mutant Q83D. (C) GDP-sugars employed in this work. GDP-D-mycosamine (10) was generated via reverse GT-catalysis, GDP-D-glucose (18), GDP-D-mannose (19) and GDP-L-fucose (22) were commercially available; GDP-L-gulose (17), GDP-D-altrose (20), GDP-D-talose (21) and GDP-L-mannose (23) were chemically synthesized (scheme A) and GDP-sugars 24–35 were enzymatically synthesized (scheme B).