Pseudoglycosyltransferase catalyzes nonglycosidic C-N coupling in validamycin a biosynthesis

Abstract

Glycosyltransferases are ubiquitous in nature. They catalyze a glycosidic bond formation between sugar donors and sugar or nonsugar 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 'pseudoglycosyltransferase' 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.

Figure 1

Chemical structures of validamycin A, acarbose, and related natural products

Figure 2

Biosynthetic gene clusters for validamycin A and characterization of VldB. A, Genetic organizations of the validamycin biosynthetic gene clusters from S. hygroscopicus 5008 and S. hygroscopicus subsp. limoneus; B, SDS-PAGE of recombinant VldB, VldE, VldH, OtsA (E. coli trehalose 6-phosphate synthase), and OtsB (E. coli trehalose 6-phosphate phosphatase) purified by Ni-NTA affinity chromatography; C, Relative specificity for nucleotidyl donors of VldB in the presence of 1 mM and 10 mM Mg²⁺.

Figure 3

HPLC and ESI-MS analyses of VldB, VldE, and VldH reaction products. A, HPLC traces of reaction mixture containing VldB, valienol 1-phosphate, and GTP. GDP-valienol (cyan block arrow) eluted in two tautomeric forms; B, HPLC trace of reaction mixture containing VldB, VldE, valienol 1-phosphate, GTP, and validamine 7-phosphate; C, HPLC trace of authentic (synthetically prepared) validoxylamine A 7′-phosphate; D, HPLC trace of reaction mixture containing VldB, VldE, VldH, valienol 1-phosphate, GTP, and validamine 7-phosphate; E, HPLC trace of authentic validoxylamine A; F, (−)ESIMS of reaction mixture containing VldB, valienol 1-phosphate, and GTP; G, (+)ESIMS of reaction mixture containing VldB, VldE, valienol 1-phosphate, GTP, and validamine 7-phosphate; H, (+)ESIMS of authentic validoxylamine A 7′-phosphate; I, (+)ESIMS of reaction mixture containing VldB, VldE, VldH, valienol 1-phosphate, GTP, and validamine 7-phosphate; J, (+)ESIMS of authentic validoxylamine A; K, ESI-MS/MS of GDP-valienol (m/z 600, cyan block arrow) from F; L, ESI-MS/MS of validoxylamine A 7′-phosphate (m/z 416, red block arrow) from G; M, ESI-MS/MS of authentic validoxylamine A 7′-phosphate; N, ESI-MS/MS of validoxylamine A (m/z 336, green block arrow) from I; O, ESI-MS/MS of authentic validoxylamine A. Yellow circle, GTP; Open circle, GDP.

Figure 4

Kinetic studies of VldE. A, Michaelis-Menten curve for validamine 7-phosphate; B, Michaelis-Menten curve for GDP-valienol.

Figure 5

Proposed catalytic mechanism for VldE and its similarity to that of farnesyl diphosphate (FPP) synthase and trehalose 6-phosphate synthase (OtsA). A, Catalytic mechanism of FPP synthases; B, Proposed S_Ni-like mechanism for OtsA; C, Proposed catalytic mechanism of VldE adopting those of FPP synthase and OtsA.

Scheme 1

Synthesis of validamine (16) and validamine 7-phosphate (21)

Scheme 2

Biosynthetic pathway to validamycin A and characterization of VldB, VldE, and VldH.

Scheme 3

Synthesis of validoxylamine A 7′-phosphate (26).