Improved one-pot multienzyme (OPME) systems for synthesizing UDP-uronic acids and glucuronides

Abstract

Arabidopsis thaliana glucuronokinase (AtGlcAK) was cloned and shown to be able to use various uronic acids as substrates to produce the corresponding uronic acid-1-phosphates. AtGlcAK or Bifidobacterium infantis galactokinase (BiGalK) was used with a UDP-sugar pyrophosphorylase, an inorganic pyrophosphatase, with or without a glycosyltransferase for highly efficient synthesis of UDP-uronic acids and glucuronides. These improved cost-effective one-pot multienzyme (OPME) systems avoid the use of nicotinamide adenine dinucleotide (NAD(+))-cofactor in dehydrogenase-dependent UDP-glucuronic acid production processes and can be broadly applied for synthesizing various glucuronic acid-containing molecules.

Scheme 1

A one-pot multienzyme (OPME) strategy for chemoenzymatic synthesis of uronosides. GlyK, glycokinase; USP, UDP-sugar pyrophosphorylase; PmPpA, Pasteurella multocida inorganic pyrophosphatase; UAT, uronosyltransferase. D-GlcA (1), GlcA-1-P (5), and UDP-GlcA (9): R¹ = R³ = OH, R² = R⁴ = R⁶ = H, R⁵ = CO₂H; D-GalA (2), GalA-1-P (6), and UDP-GalA (10): R¹ = R⁴ = OH, R² = R³ = R⁶ = H, R⁵ = CO₂H; D-ManA (3), ManA-1-P (7), and UDP-ManA (11): R² = R³ = OH, R¹ = R⁴ = R⁶ = H, R⁵ = CO₂H; L-IdoA (4), IdoA-1-P (8), and UDP-IdoA (12): R¹ = R³ = OH, R² = R⁴ = R⁵ = H, R⁶ = CO₂H.

Scheme 2

Sequential OPME synthesis of heparosan disaccharide (14), trisaccharide (15), and tetrasaccharide (16). Enzymes used: NahK, N-acetylhexosamine-1-phosphate kinase; PmGlmU, Pasteurella multocida N-acetylglucosamine-1-phosphate uridylyltransferase; PmPpA, Pasteurella multocida inorganic pyrophosphatase; PmHS2, Pasteurella multocida heparosan synthase 2; AtGlcAK, Arabidopsis thaliana glucuronokinase; BLUSP, Bifidobacterium longum UDP-sugar pyrophosphorylase.