title: [Identification of the catalytic residues of mannanase from Alicyclobacillus acidocaldarius].
abstract: OBJECTIVE
  To identify the catalytic residues of mannanase AaManA from Alicyclobacillus acidocaldarius.
  
  
  METHODS
  Based on the sequence alignment by ClustalX and ESPript and the structure information of GH -53 family, the possible catalytic residues were selected and mutated by overlap extension PCR. The protein of wild type and mutant were expressed in E. coli BL21 (DE3) and ordinal purified by Ni - NTA affinity chromatography, gel - filtrate chromatography and ion - exchange chromatography. The purified protein was analyzed by thin layer chromatography (TLC) and the dinitrosalicylic acid (DNS) methods for enzyme assay.
  
  
  RESULTS
  Seven mutants, E151A, E159A, E231A, C150A, E151Q, E231Q and double mutation E151Q&E231Q were successful constructed. Mutant E159A showed similar activities with wild type, and C150A mutation resulted in only a 3 -fold reduction in the activities, but mutations E151A, E231A, E151Q, E231Q and E151Q&E231Q resulted in sharp decreases or loss in the activities, indicating that Glu151 and Glu231 play critical roles in AaManA activity. Furthermore, the presence of Glu151 at the C terminus of beta4 and Glu231 at the C terminus of beta7 was entirely consistent with the positions of the acid/base catalyst and the nucleophile catalyst of a GH - A enzyme, respectively.
  
  
  CONCLUSIONS
  By combining the results of TLC and enzyme assay of those mutants and the structural comparisons, it was confirmed that Glu151 and Glu231 fulfilled the roles of an acid/base catalyst and nucleophile catalyst in AaManA, respectively.
authors.affiliation: College of Trouism, Hebei Normal University, Shijiazhuang 050016, China. shujingxu@163.com
authors.forename: Shujing
authors.initials: S
authors.lastname: Xu
authors.forename: Caifeng
authors.initials: C
authors.lastname: Zhang
authors.forename: Zhangwei
authors.initials: Z
authors.lastname: Xue
authors.forename: Guangzheng
authors.initials: G
authors.lastname: He
authors.forename: Jiansong
authors.initials: J
authors.lastname: Ju
authors.forename: Baohua
authors.initials: B
authors.lastname: Zhao
mesh_terms: D056450:Alicyclobacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D044902:beta-Mannosidase