title: Switching substrate preference of thermophilic xylose isomerase from D-xylose to D-glucose by redesigning the substrate binding pocket.
abstract: The substrate specificity of thermophilic xylose isomerase from Clostridium thermosulfurogenes was examined by using predictions from the known crystal structure of the Arthrobacter enzyme and site-directed mutagenesis of the thermophile xylA gene. The orientation of glucose as a substrate in the active site of the thermophilic enzyme was modeled to position the C-6 end of hexose toward His-101 in the substrate-binding pocket. The locations of Met-87, Thr-89, Val-134, and Glu-180, which contact the C-6-OH group of the substrate in the sorbitol-bound xylose isomerase from Arthrobacter [Collyer, C.A., Henrick, K. & Blow, D. M. (1990) J. Mol. Biol. 212, 211-235], are equivalent to those of Trp-139, Thr-141, Val-186, and Glu-232 in the thermophilic enzyme. Replacement of Trp-139 with Phe reduced the Km and enhanced the kcat of the mutant thermophilic enzyme toward glucose, whereas this substitution reversed the effect toward xylose. Replacement of Val-186 with Thr also enhanced the catalytic efficiency of the enzyme toward glucose. Double mutants with replacements Trp-139----Phe/Val-186----Thr and Trp-139----Phe/Val-186----Ser had a higher catalytic efficiency (kcat/Km) for glucose than the wild-type enzyme of 5- and 2-fold, respectively. They also exhibited 1.5- and 3-fold higher catalytic efficiency for D-glucose than for D-xylose, respectively. These results provide evidence that alteration in substrate specificity of factitious thermophilic xylose isomerases can be achieved by designing reduced steric constraints and enhanced hydrogen-bonding capacity for glucose in the substrate-binding pocket of the active site.
authors.affiliation: Michigan Biotechnology Institute, Lansing 48909.
authors.forename: M
authors.initials: M
authors.lastname: Meng
authors.forename: C
authors.initials: C
authors.lastname: Lee
authors.forename: M
authors.initials: M
authors.lastname: Bagdasarian
authors.forename: J G
authors.initials: JG
authors.lastname: Zeikus
mesh_terms: D019747:Aldose-Ketose Isomerases; D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology*; D003198:Computer Simulation; D004252:DNA Mutational Analysis; D005947:Glucose / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D014994:Xylose / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014994:Xylose; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D005947:Glucose