Increased D-allose production by the R132E mutant of ribose-5-phosphate isomerase from Clostridium thermocellum.
Ribose-5-phosphate isomerase from Clostridium thermocellum converted D-psicose to D-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in D-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for D-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency (k(cat)/K(m)) of the R132E mutant for D-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of D-psicose to D-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min.
Department of Bioscience and Biotechnology, Konkuk University, Seoul, Republic of Korea.
Soo-Jin
SJ
Yeom
Eun-Sun
ES
Seo
Yeong-Su
YS
Kim
Deok-Kun
DK
Oh
D019747:Aldose-Ketose Isomerases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D020134:Catalytic Domain; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D005632:Fructose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D013696:Temperature
C003243:psicose; D005632:Fructose; C002055:allose; D019747:Aldose-Ketose Isomerases; C022548:ribosephosphate isomerase; D005947:Glucose