Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose

Abstract

Derivatives of 3-amino-3,6-dideoxyhexoses are widespread in Nature. They are part of the repeating units of lipopolysaccharide O-antigens, of the glycan moiety of S-layer (bacterial cell surface layer) glycoproteins and also of many antibiotics. In the present study, we focused on the elucidation of the biosynthesis pathway of dTDP-alpha-D-Quip3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose) from the Gram-positive, anaerobic, thermophilic organism Thermoanaerobacterium thermosaccharolyticum E207-71, which carries Quip3NAc in its S-layer glycan. The biosynthesis of dTDP-alpha-D-Quip3NAc involves five enzymes, namely a transferase, a dehydratase, an isomerase, a transaminase and a transacetylase, and follows a pathway similar to that of dTDP-alpha-D-Fucp3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-galactose) biosynthesis in Aneurinibacillus thermoaerophilus L420-91(T). The ORFs (open reading frames) of interest were cloned, overexpressed in Escherichia coli and purified. To elucidate the enzymatic cascade, the different products were purified by HPLC and characterized by NMR spectroscopy. The initiating reactions catalysed by the glucose-1-phosphate thymidylyltransferase RmlA and the dTDP-D-glucose-4,6-dehydratase RmlB are well established. The subsequent isomerase was shown to be capable of forming a dTDP-3-oxo-6-deoxy-D-glucose intermediate from the RmlB product dTDP-4-oxo-6-deoxy-D-glucose, whereas the isomerase involved in the dTDP-alpha-D-Fucp3NAc pathway synthesizes dTDP-3-oxo-6-deoxy-D-galactose. The subsequent reaction steps of either pathway involve a transaminase and a transacetylase, leading to the specific production of nucleotide-activated 3-acetamido-3,6-dideoxy-alpha-D-glucose and 3-acetamido-3,6-dideoxy-alpha-D-galactose respectively. Sequence comparison of the ORFs responsible for the biosynthesis of dTDP-alpha-D-Quip3NAc revealed homologues in Gram-negative as well as in antibiotic-producing Gram-positive bacteria. There is strong evidence that the elucidated biosynthesis pathway may also be valid for LPS (lipopolysaccharide) O-antigen structures and antibiotic precursors.

Figure 1. Chromosomal organization of the genes encoding dTDP-α-d-Quip3NAc and dTDP-β-l-rhamnose biosynthesis in T. thermosaccharolyticum E207-71 (partial slg gene cluster; GenBank^® accession number AY422724)

Figure 2. Comparison of the biosynthesis pathways for dTDP-α-d-Quip3NAc and dTDP-α-d-Fucp3NAc

(I) d-Glucose-1-phosphate; (II) dTDP-d-glucose; (III) dTDP-4-oxo-6-deoxy-d-glucose; (IV) dTDP-3-oxo-6-deoxy-d-glucose; (V) dTDP-d-Quip3N; (VI) dTDP-d-Quip3NAc; (VII) dTDP-3-oxo-6-deoxy-d-galactose; (VIII) dTDP-d-Fucp3N, (IX) dTDP-d-Fucp3NAc. RmlA, glucose-1-phosphate thymidylyltransferase; RmlB, dTDP-d-glucose 4,6-dehydratase; QdtA, dTDP-4-oxo-6-deoxy-d-glucose-3,4-oxoisomerase; QdtB, dTDP-3-oxo-6-deoxy-d-glucose aminase; QdtC, dTDP-d-Quip3N acetylase; FdtA, dTDP-4-oxo-6-deoxy-d-glucose-3,4-oxoisomerase; FdtB, dTDP-3-oxo-6-deoxy-d-galactose aminase; FdtC, dTDP-d-Fucp3N acetylase.

Figure 3. HPLC analysis of the products of the enzymatic reactions catalysed by QdtA, QdtB and QdtC

(A) dTDP-glucose II; (B) dTDP-4-oxo-6-deoxy-d-glucose III, synthesized from II by RmlB; (C) dTDP-d-Quip3N V, synthesized from III by sequential action of QdtA and QdtB; (D) dTDP-d-Quip3NAc VI, final reaction product of the concerted action of RmlB, QdtA, QdtB and QdtC using II as substrate. The numbers used for designation of enzymatic products are identical with those used in Figure 2.

Figure 4. 300 MHz ¹H NMR spectrum of dTDP-Quip(3)NAc VI recorded at 300 K in ²H₂O

The signal at 3.7 p.p.m. (indicated by an arrow) corresponds to residual Tris buffer; the signal at ~7.4 p.p.m. (indicated by an arrow) is due to residual imidazole.

Figure 5. Amino acid sequence alignment of T. thermosaccharolyticum E207-71 QdtA, A. thermoaerophilus L420-91^T FdtA and S. fradiae Tyl1a

The amino acids of the active site are in boldface letters and the characteristic signature sequence is highlighted in grey. The positions of the conserved histidine residues (His⁴⁹ and His⁵¹ in FdtA) are indicated by black dots. Note that His⁹⁵ (indicated by a grey dot) in FdtA is an arginine residue in QdtA and in Tyl1a.