Roles of Ile66 and Ala107 of D-psicose 3-epimerase from Agrobacterium tumefaciens in binding O6 of its substrate, D-fructose.
Using site-directed mutagenesis, we investigated the roles of Ile66 and Ala107 of D: -psicose 3-epimerase from Agrobacterium tumefaciens in binding O6 of its true substrate, D: -fructose. When Ile66 was substituted with alanine, glycine, cysteine, leucine, phenylalanine, tryptophan, tyrosine or valine, all the mutants dramatically increased the K (m) for D: -tagatose but slightly decreased the K (m) for D: -fructose, indicating that Ile66 is involved in substrate recognition. When Ala107 was substituted by either isoleucine or valine, the substituted mutants had lower thermostability than the wild-type enzyme whereas the proline-substituted mutant had higher thermostability. Thus, Ala107 is involved in enzyme stability.
Kim
Hye-Jung
HJ
Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, 143-701, South Korea.
Lim
Byung-Chul
BC
Yeom
Soo-Jin
SJ
Kim
Yeong-Su
YS
Kim
Dooil
D
Oh
Deok-Kun
DK
D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005632:Fructose / Q000378:metabolism*; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; C003243:psicose; D005632:Fructose; D019998:Racemases and Epimerases