title: Stabilized homoserine o-succinyltransferases (MetA) or L-methionine partially recovers the growth defect in Escherichia coli lacking ATP-dependent proteases or the DnaK chaperone.
abstract: BACKGROUND
  The growth of Escherichia coli at elevated temperatures is limited due to the inherent instability of homoserine o-succinyltransferase, MetA, which is the first enzyme in the methionine biosynthesis pathway. MetA is also unstable under other stressful conditions, such as weak organic acids and oxidative stress. The MetA protein unfolds, even at 25°C, forms considerable aggregates at 37°C and completely aggregates at 44°C.
  
  
  RESULTS
  We extended the MetA mutation studies using a consensus concept based on statistics and sequence database analysis to predict the point mutations resulting in increased MetA stability. In this study, four single amino acid substitutions (Q96K, I124L, I229Y and F247Y) in MetA designed according to the consensus concept and using the I-mutant2.0 modeling tool conferred accelerated growth on the E. coli strain WE at 44°C. MetA mutants that enabled E. coli growth at higher temperatures did not display increased melting temperatures (Tm) or enhanced catalytic activity but did show improved in vivo stability at mild (37°C) and elevated (44°C) temperatures. Notably, we observed that the stabilized MetA mutants partially recovered the growth defects of E. coli mutants in which ATP-dependent proteases or the DnaK chaperone was deleted. These results suggest that the impaired growth of these E. coli mutants primarily reflect the inherent instability of MetA and, thus, the methionine supply. As further evidence, the addition of methionine recovered most of the growth defects in mutants lacking either ATP-dependent proteases or the DnaK chaperone.
  
  
  CONCLUSIONS
  A collection of stable single-residue mutated MetA enzymes were constructed and investigated as background for engineering the stabilized mutants. In summary, the mutations in a single gene, metA, reframe the window of growth temperature in both normal and mutant E. coli strains.
authors.affiliation: Superbacteria Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong-gu, Daejeon 305-806, South Korea.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Mordukhova
authors.forename: Dooil
authors.initials: D
authors.lastname: Kim
authors.forename: Jae-Gu
authors.initials: JG
authors.lastname: Pan
mesh_terms: D049069:ATP-Dependent Proteases / Q000172:deficiency*; D019943:Amino Acid Substitution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development* / Q000528:radiation effects; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000172:deficiency*; D051082:Homoserine O-Succinyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism*; D020125:Mutation, Missense; D017354:Point Mutation; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D008715:Methionine; D051082:Homoserine O-Succinyltransferase; C494973:metA protein, E coli; D049069:ATP-Dependent Proteases; C063842:dnaK protein, E coli
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