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Biochemical characterization of a UDP‐sugar pyrophosphorylase from Thermus caldophilus GK24
Author(s) -
Kim Joong Su,
Koh Sukhoon,
Shin HyunJae,
Lee DaeSil,
Lee Se Yong
Publication year - 1999
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1999.tb01143.x
Subject(s) - thermus , enzyme , chemistry , biochemistry , size exclusion chromatography , chromatography , molecular mass , sugar , fructose , specific activity , ion chromatography , enzyme assay , sugar phosphates , thermophile
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 Mg 2+ 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.