Characterization of a Mannose-6-Phosphate Isomerase from Thermus thermophilus and Increased l -Ribose Production by Its R142N Mutant

Abstract

ABSTRACT An uncharacterized gene from Thermus thermophilus , thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli . The maximal activity of the recombinant enzyme for l -ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu 2+ . Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of l -ribulose to l -ribose, a potential starting material for many l -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 in l -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 ( k cat / K m ) for l -ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The k cat / K m of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported k cat / K m . The R142N mutant enzyme produced 213 g/liter l -ribose from 300 g/liter l -ribulose for 2 h, with a volumetric productivity of 107 g liter −1 h −1 , which was 1.5-fold higher than that of the wild-type enzyme.