Probing the molecular determinant for the catalytic efficiency of L-arabinose isomerase from Bacillus licheniformis.
Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of 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 to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (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, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.
Prabhu
Ponnandy
P
Department of Bioscience and Biotechnology, Institute of Biomedical Science and Technology, South Korea.
Jeya
Marimuthu
M
Lee
Jung-Kul
JK
D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D001426:Bacterial Proteins; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase