Chemoenzymatic Synthesis and Applications of Prokaryote-Specific UDP-Sugars

Abstract

This method describes the chemoenzymatic synthesis of several nucleotide sugars, which are essential substrates in the biosynthesis of prokaryotic N- and O-linked glycoproteins. Protein glycosylation is now known to be widespread in prokaryotes and proceeds via sequential action of several enzymes, utilizing both common and modified prokaryote-specific sugar nucleotides. The latter, which include UDP-hexoses such as UDP-diNAc-bacillosamine (UDP-diNAcBac), UDP-diNAcAlt, and UDP-2,3-diNAcManA, are also important components of other bacterial and archaeal glycoconjugates. The ready availability of these "high-value" intermediates will enable courses of study into inhibitor screening, glycoconjugate biosynthesis pathway discovery, and unnatural carbohydrate incorporation toward metabolic engineering.

Keywords: 2,3-di-N-acetyl-glucuronic acid; 2,4-di-N-acetyl-bacillosamine; Chemoenzymatic synthesis; Glycoconjugate biosynthesis; Nucleotide-activated carbohydrates; Pseudaminic acid.

Fig. 1

Structures of selected prokaryote-specific glycosyl donors. Shown are donors from Pseudomonas aeruginosa (P. aeruginosa); Bordatella pertussis, (B. pertussis); Campylobacter jejuni, (C. jejuni); Vibrio cholera, (V. cholera); Mycobacterium tuberculosis, (M. tuberculosis); Klebsiella pneumoniae, (K. pneumonia); Helicobacter pylori, (H. pylori); and Legionella pneumoniae, (L. pneumonia).

Fig. 2

N- and O-linked prokaryotic glycans featuring modified carbohydrate building blocks.

Fig. 3

Biosynthetic pathway of UDP-diNAcBacillosamine in C. jejuni.

Fig. 4

FPLC profile (280 nm, 1.5 mL/min, y-axis in volume) from chemoenzymatic synthesis of UDP-4-aminosugar.

Fig. 5

CMP-pseudaminic acid biosynthetic pathway. Steps covered in this method are boxed.

Fig. 6

NP-HPLC profiles (260 nm, 1 mL/min, y-axis in volume) from chemoenzymatic synthesis of UDP-6-deoxy-4-amino-2-NAc-L-Alt. a) Reaction at t = 0; b) Reaction progress; c) Purified UDP-6-deoxy-4-amino-2-NAc-L-Alt (3).

Fig. 7

Steady State kinetics for C. jejuni acetyltransferase PseH.

Fig. 8

Biosynthetic pathway of UDP-2,3-diNAcManA, a lipopolysaccharide subunit in P. aeruginosa PAO1. UDP-2,3-diNAcGlcA is boxed.

Fig. 9

Capillary electrophoresis time course analysis of the WbpD reaction. (1) AcCoA; (2) CoASH; (3) UDP-2,3-diNAcGlcA; (4) UDP-2-NAc-3-NH₂GlcA. The peak labeled x represents an impurity present in the AcCoA starting material (Larkin & Imperiali 2009).