Biochemical characterization of a UDP‐sugar pyrophosphorylase from Thermus caldophilus GK24

Abstract

An extremely thermostable UDP‐GlcNAc pyrophosphorylase has been purified from Thermus caldophilus GK24 by chromatographic methods including ion‐exchange, hydrophobic interaction, and affinity chromatographies. The specific activity of the enzyme was enriched 41.8‐fold, with a recovery of 2%. The molecular mass of the enzyme was 41 kDa by SDS/PAGE and 45 kDa by gel‐filtration chromatography. The activity was maximum at 86 °C and its half‐life at 95 °C was 30 min. Its optimum pH was 6.9 in the presence of Mg2+ ions. A biochemical study showed that UDP‐GlcNAc pyrophosphorylase activity could be enhanced by fructose 1‐phosphate, a precursor of UDP‐GlcNAc. The enzyme showed a broad substrate specificity with sugar 1‐phosphates, including glucose 1‐phosphate, GlcNAc‐1‐P and xylose 1‐phosphate. The enzyme was therefore named UDP‐sugar pyrophosphorylase. The N‐terminal and internal peptide sequences were determined and compared with known sequences from various sources. It was found that N‐terminal sequence is similar to that of UDP‐GlcNAc and UDP‐glucose pyrophosphorylases from other bacterial sources.

Engineering a Robust UDP-Glucose Pyrophosphorylase for Enhanced Biocatalytic Synthesis via ProteinMPNN and Ancestral Sequence Reconstruction

“…The other method involves using glucose-1-phosphate (Glc-1P) as a substrate, consuming UTP, and under the catalysis of UDP-Glc pyrophosphorylase (UGP, EC 2.7.7.9), synthesizing UDP-Glc . However, despite the discovery of UGP over half a century ago and the demonstrated activity of numerous UGPs from thermophilic bacteria, thermophilic archaea, or neutrophilic bacteria, it is still rarely used as a biocatalyst for UDP-Glc production. − This is due to the high cost of the key raw material, Glc-1P, in this method. Additionally, the relatively poor thermal stability of UGP limits the industrial-scale application of this method.…”