Table 1

Oligonucleotide primers for mutagenesis and enzyme activity of mutants

Mutation*Primer (5′ to 3′)Relative activity, %
H104    H106    D108 H109
Wild type ATTATTAGTTCT CAC TTG CAT TTT GAT  CAT GCAGGAGGAAATGGC 100.0
H104S/H106S/D108S/H109S ATTATTAGTTCT TCC TTG TCT TTT TCT  TCT GCAGGAGGAAATGGC 0.0
H104S/H106S/H109S ATTATTAGTTCT TCC TTG TCT TTT GAT  TCT GCAGGAGGAAATGGC 0.0
D108S/H109S ATTATTAGTTCT CAC TTG CAT TTT CTT  CTT GCAGGAGGAAATGGC 0.0
H104S/H106S ATTATTAGTTCT TCC TTG TCT TTT GAT  CAT GCAGGAGGAAATGGC 51.1
H104L/H106L/D108L/H109L ATTATTAGTTCT CTC TTG CTT TTT CTT  CTT GCAGGAGGAAATGGC 37.9
H104L/H106L/D108L ATTATTAGTTCT CTC TTG CTT TTT CTT  CAT GCAGGAGGAAATGGC 61.4
H104S ATTATTAGTTCT TCC TTG CAT TTT GAT  CAT GC 100.0
H106S ATTATTAGTTCT CAC TTG TCT TTT GAT  CAT GC 61.4
D108S           CT CAC TTG CAT TTT TCT  CAT GCAGGAGGAAATGGC 0.0
D108E           CT CAC TTG CAT TTT GAA  CAT GCAGGAGGAAATGGC 90.8
H109S           CT CAC TTG CAT TTT GAT  TCT GCAGGAGGAAATGGC 0.0
H169
Wild type GCATACACCAGGC CAT ACTCCAGGGCATCAATCG 100.0
H169S GCATACACCAGGC TCT ACTCCAGGGCATCAATCG 61.4
  • * The numbers indicate the position of the target amino acids in AiiA peptide; the letters before and after numbers indicate, respectively, the original and substitute amino acids. 

  • The corresponding conserved amino acids and their positions in AiiA peptide are marked above the primer sequences. Spaces were introduced in the primer sequences for the convenience of inspection. The bases changed were printed boldface and underlined in each primer. 

  • The enzyme bioassay results of each mutant are expressed as activity relative to that of wild-type AiiA; bioassay was repeated three times, and data are means of two replicates.