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Journal of Bacteriology, May 2007, p. 3556-3563, Vol. 189, No. 9
0021-9193/07/$08.00+0     doi:10.1128/JB.01826-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Probing the Essential Catalytic Residues and Substrate Affinity in the Thermoactive Bacillus stearothermophilus US100 L-Arabinose Isomerase by Site-Directed Mutagenesis

Laboratoire d'Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax BP "K," 3038 Sfax, Tunisie,¹ Laboratoire de BioCristallographie, Institut de Biologie et Chimie des Protéines, UMR 5086-CNRS/Université de Lyon 1, IFR128 "BioSciences Lyon-Gerland," 7 Passage du Vercors, F-69367 Lyon Cedex 07, France²

Received 5 December 2006/ Accepted 19 February 2007

The L-arabinose isomerase (L-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of L-arabinose isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 L-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 L-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the L-fucose isomerase and the L-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 L-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 L-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for L-fucose and decreased the affinity for L-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.

Published ahead of print on 2 March 2007.