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Yeom, Soo-Jin,Kim, Yeong-Su,Oh, Deok-Kun American Society for Microbiology 2013 Applied and environmental microbiology Vol.79 No.3
<B>ABSTRACT</B><P>Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase fromClostridium difficile(CDRPI), mannose-6-phosphate isomerase fromBacillus subtilis(BSMPI), and glucose-6-phosphate isomerase fromPyrococcus furiosus(PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphate-binding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to ad-ribose isomerase, anl-ribose isomerase, and anl-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases.</P>
Yeom, Soo-Jin,Seo, Eun-Sun,Kim, Bi-Na,Kim, Yeong-Su,Oh, Deok-Kun American Society for Microbiology 2011 Applied and environmental microbiology Vol.77 No.3
<B>ABSTRACT</B><P>An uncharacterized gene from<I>Thermus thermophilus</I>, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in<I>Escherichia coli</I>. The maximal activity of the recombinant enzyme forl-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu<SUP>2+</SUP>. Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion ofl-ribulose tol-ribose, a potential starting material for manyl-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase inl-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (<I>k</I>cat/<I>Km</I>) forl-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The<I>k</I>cat/<I>Km</I>of the R142N mutant was 3.8-fold higher than that of<I>Geobacillus thermodenitrificans</I>mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported<I>k</I>cat/<I>Km</I>. The R142N mutant enzyme produced 213 g/literl-ribose from 300 g/literl-ribulose for 2 h, with a volumetric productivity of 107 g liter<SUP>−1</SUP>h<SUP>−1</SUP>, which was 1.5-fold higher than that of the wild-type enzyme.</P>