Title: Tryptophan 266 is Required for the C-C Hydrolase Activity
Abstract: As a member of the α/β-hydrolase superfamily,Rhodococcus sp.R04 derived BphD C-C hydrolase(2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate,HOPDA hydrolase) catalyses the hydrolytic C-C cleavage of the meta-ring fission products from the biphenyl catabolic pathway.We constructed six mutants of C-C hydrolase to investigate its catalytic mechanism.Substitutions of Ser-110,Asp-237 and His-265 in HOPDA hydrolase with Ala caused to 17~19-fold reduction in kcat,and reduced the kcat/Km values by 104~103 folds in comparison to the wild-type.The Ala substitution of Trp-85 and Trp-219 led to 3.6-and 3.3-fold reduction in kcat/Km,respectively.Mutating of Trp-266 to Ala resulted in a 104-fold reduction of kcat/Km.From the circular dichroism(CD)spectra,S110A,D237A,H265A and W266A were predominately showed in α-helix conformation as the wild-type.The chemical modification results showed that the modifiers did not influence the activities of S110A,D237A,H265A and Trp266 mutants,except 0.5 mmol/L NBS reduced the activity of W85A by 90% and W219A by70%.Our data indicated that Ser110,Asp237 and His265 were necessary for the catalytic reaction of HOPDA hydrolase,and Trp266 was suggested to play a key role in the enzymatic reaction.
Publication Year: 2012
Publication Date: 2012-01-01
Language: en
Type: article
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Cited By Count: 1
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