title: Roles of tryptophan residue and disulfide bond in the variable lid region of oxidized polyvinyl alcohol hydrolase.
abstract: Oxidized polyvinyl alcohol hydrolase (OPH) catalyzes the cleavage of C-C bond in β-diketone. It belongs to the α/β-hydrolase family and contains a unique lid region that covers the active site. The lid is the most variable region when pOPH from Pseudomonas sp. VM15C and sOPH from Sphingopyxis sp. 113P3 are compared. The wild-type enzymes and the pOPH mutants W255A, W255Y and W255F were analyzed for lipase activity by using p-nitrophenyl (pNP) esters as the substrates. The wild-type enzymes showed increased Km and decreased kcat/Km with the acyl chain length, and the mutants showed reduced kcat/Km for pNP acetate, indicating the importance of Trp255 in sequestering the active site from solvent. The significantly lower activity for pNP butyrate can be a result of product inhibition, as suggested by the complex crystal structures, in which butyric acid, DMSO or PEG occupied the same substrate-binding cleft. The mutant activity was retained with pNP caprylate and pNP laurate as the substrates, reflecting the amphipathic nature of the cleft. Moreover, the disulfide bond formation of Cys257/267 is important for the activity of pOPH, but it is not essential for sOPH, which has a shorter lid structure.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Tzu-Ping
authors.initials: TP
authors.lastname: Ko
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Chun-Hsiang
authors.initials: CH
authors.lastname: Huang
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. Electronic address: guo_rt@tib.cas.cn.
authors.forename: Rey-Ting
authors.initials: RT
authors.lastname: Guo
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China. Electronic address: gcdu@jiangnan.edu.cn.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002087:Butyrates / Q000737:chemistry / Q000378:metabolism; D002210:Caprylates / Q000737:chemistry / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D007848:Laurates / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011549:Pseudomonas / Q000737:chemistry* / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020578:Sphingomonas / Q000737:chemistry* / Q000201:enzymology; D055672:Static Electricity; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002087:Butyrates; D002210:Caprylates; D004220:Disulfides; D007848:Laurates; D011994:Recombinant Proteins; C034106:4-nitrophenyloctanoate; C050014:4-nitrophenyl laurate; C033592:4-nitrophenyl butyrate; D014364:Tryptophan; D002265:Carboxylic Ester Hydrolases; C409492:oxidized polyvinyl alcohol hydrolase, Pseudomonas sp.
keywords: Crystal structure; Enzyme kinetics; Lipase activity; Mutagenesis; p-Nitrophenyl esters