title: Characterization of a F280N variant of L-arabinose isomerase from Geobacillus thermodenitrificans identified as a D-galactose isomerase.
abstract: The double-site variant (C450S-N475K) L-arabinose isomerase (L-AI) from Geobacillus thermodenitrificans catalyzes the isomerization of D-galactose to D-tagatose, a functional sweetener. Using a substrate-docking homology model, the residues near to D-galactose O6 were identified as Met186, Phe280, and Ile371. Several variants obtained by site-directed mutagenesis of these three residues were analyzed, and a triple-site (F280N) variant enzyme exhibited the highest activity for D-galactose isomerization. The k cat/K m of the triple-site variant enzyme for D-galactose was 2.1-fold higher than for L-arabinose, whereas the k cat/K m of the double-site variant enzyme for L-arabinose was 43.9-fold higher than for D-galactose. These results suggest that the triple-site variant enzyme is a D-galactose isomerase. The conversion rate of D-galactose to D-tagatose by the triple-site variant enzyme was approximately 3-fold higher than that of the double-site variant enzyme for 30 min. However, the conversion yields of L-arabinose to L-ribulose by the triple-site and double-site variant enzymes were 10.6 and 16.0 % after 20 min, respectively. The triple-site variant enzyme exhibited increased specific activity, turnover number, catalytic efficiency, and conversion rate for D-galactose isomerization compared to the double-site variant enzyme. Therefore, the amino acid at position 280 determines the substrate specificity for D-galactose and L-arabinose, and the triple-site variant enzyme has the potential to produce D-tagatose on an industrial scale.
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, 143-701, South Korea.
authors.forename: Baek-Joong
authors.initials: BJ
authors.lastname: Kim
authors.forename: Seung-Hye
authors.initials: SH
authors.lastname: Hong
authors.forename: Kyung-Chul
authors.initials: KC
authors.lastname: Shin
authors.forename: Ye-Seul
authors.initials: YS
authors.lastname: Jo
authors.forename: Deok-Kun
authors.initials: DK
authors.lastname: Oh
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D001089:Arabinose / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D056506:Geobacillus / Q000201:enzymology* / Q000235:genetics; D006601:Hexoses / Q000378:metabolism; D007535:Isomerases; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D013379:Substrate Specificity
chemical_list: D006601:Hexoses; D050505:Mutant Proteins; D001089:Arabinose; D007535:Isomerases; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase; C030192:tagatose; D005690:Galactose