P76: Probing the Molecular Determinant for Catalytic Efficiency of L-Arabinose Isomerase from Bacillus licheniformis

Bacillus licheniformis L-arabinose isomerase (BLAI) is distinguished from other L-AIs by its high substrate specificity for L-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis of individual screened residues, was used to address the molecular determinants for the catalytic efficiency of BLAI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type BLAI was identified as an important residue affecting the catalytic efficiency of BLAI. Further insights into the function of residue Y333 were obtained by substituting it with other aromatic, non-polar hydrophobic amino acids or polar amino acids. Substituting Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward L-arabinose. In contrast, the activity of mutants containing hydrophobic amino acids (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, or Lys, showed almost no activity with L-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in catalytic efficiency of BLAI.