title: [Construction and characterization of R169H mutant of aspartokinase from Corynebacterium pekinense].
abstract: OBJECTIVE
  Increasing the activity of aspartokinase (AK) from Corynebacterium pekinense.
  
  
  METHODS
  The gene of AK was constructed and mutated by site-specific mutagenesis. The mutational recombinant plasmid was heterologously expressed in Escherichia coli BL21. The mutational AK was purified by Ni(2+)-NTA column after ultrasonicating of the recombinant bacteria, and then identified by SDS-PAGE and Western blot. We compared the kinetic difference between R169H AK and WT AK by determining the enzymatic activities. Some other characteristics of R169H AK and WTAK were also studied.
  
  
  RESULTS
  The mutant R169H was successfully constructed. The molecular weight of AK was 48kDa. V(max) of R169H AK was 226.3 U/mg x s(-1), which was 2.3 times higher than that of WT AK. The optimum reaction temperature of R169H AK was 26 degrees C, the same as that of WT AK. The optimum reaction pH of R169H AK was 9.0, slightly higher than that of WT AK. The half-life period of R169H AK under optimum temperature and pH were 5.5h, much higher than that of WT AK. Lysine, threonine and methionine had an active effect on the activity of R169H AK when they were in low concentration.
  
  
  CONCLUSIONS
  The hydrogen bond between R169 and E92 was broken down in R169H AK, which could affect the degree of polymerization and further lowered the affinity of mutant AK with substrates and then decreased the inhibition inducing by the metabolites. Thus, the V(max) of mutant AK from R169H had increased by 2.3 times compared with that of WT AK.
authors.forename: Huiying
authors.initials: H
authors.lastname: Li
authors.forename: Yunming
authors.initials: Y
authors.lastname: Zhu
authors.forename: Weihong
authors.initials: W
authors.lastname: Min
authors.forename: Dongling
authors.initials: D
authors.lastname: Zhan
authors.forename: Jun
authors.initials: J
authors.lastname: Ren
mesh_terms: D001222:Aspartate Kinase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003352:Corynebacterium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D020125:Mutation, Missense
chemical_list: D001426:Bacterial Proteins; D001222:Aspartate Kinase