Journal
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
Volume 85, Issue 6, Pages 1146-1158Publisher
WILEY
DOI: 10.1002/prot.25279
Keywords
chorismatase; chorismate; Fkbo; Hyg5; reaction mechanism; QM/MM
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Funding
- Natural Science Foundation of Shandong Province [ZR2015CL017]
- Natural Science Foundation of China [21573127, 21373125]
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Chorismatase is an important enzyme involved in Shikimate pathway, which catalyzes the conversion of chorismate into pyruvate and (dihydro)-benzoic acid derivatives. According to the outcomes of catalytic reactions, chorismatases can be divided into three subfamilies: CH-Fkbo, CH-Hyg5 and CH-XanB2. Recently, the crystal structures of CH-Fkbo and CH-Hyg5 from Streptomyces hygroscopicus have been successfully obtained, allowing us to perform QM/MM calculations to explore the reaction details. Our calculation results support the proposal that CH-Fkbo and CH-Hyg5 employ different catalytic mechanisms and gave the mechanistic details. Fkbo follows a typical hydrolytic mechanism, which contains three consecutive steps, including the protonation step of the methylene group of substrate, the nucleophilic attack of the resulted carbocation by activated water and cleavage of C2'-O8 bond of tetrahedral intermediate (hemiketal). The protonation of methylene group and the C2'-O8 cleavage correspond to similar energy barriers (26.5 and 24.8 kcal/mol), suggesting both steps to be rate-limiting. Whereas Hyg5 employs an intramolecular mechanism, in which the oxygen from C4 migrates to C3 via an arene oxide intermediate. The first step of Hyg5, which corresponds to the concerted protonation of methylene group and the cleavage of C3-O8, is calculated to be rate-limiting with an energy barrier of 26.3 kcal/mol. The nonconserved active site residue G240(Hyg5) (or A244(Fkb alpha)) is suggested to be responsible for leading to different reaction mechanism in CH-Fkbo and CH-Hyg5. During the catalytic reaction, residue C327 plays an important role in directing the product selectivity in Hyg5 enzyme. (C) 2017 Wiley Periodicals, Inc.
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