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.
journal_name
Proc Natl Acad Sci U S Aauthors
Meng M,Lee C,Bagdasarian M,Zeikus JGdoi
10.1073/pnas.88.9.4015subject
Has Abstractpub_date
1991-05-01 00:00:00pages
4015-9issue
9eissn
0027-8424issn
1091-6490journal_volume
88pub_type
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