Site‐directed mutagenesis of the amino acid residues in β‐strand III [Val<sup>30</sup>‐Val<sup>36</sup>] of <scp>d</scp>‐amino acid aminotransferase of <i>Bacillus</i> sp. YM‐1

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<jats:p>The β‐strand III formed by amino acid residues Val<jats:sup>30</jats:sup>‐Val<jats:sup>36</jats:sup> is located across the active site of the thermostable <jats:sc>d</jats:sc>‐amino acid aminotransferase (<jats:sc>d</jats:sc>‐AAT) from thermophilic <jats:italic>Bacillus</jats:italic> sp. YM‐1, and the odd‐numbered amino acids (Tyr<jats:sup>31</jats:sup>, Val<jats:sup>33</jats:sup>, Lys<jats:sup>35</jats:sup>) in the strand are revealed to be directed toward the active site. Interestingly, Glu<jats:sup>32</jats:sup> is also directed toward the active site. We first investigated the involvement of these amino acid residues in catalysis by alanine scanning mutagenesis. The Y31A and E32A mutant enzymes showed a marked decrease in <jats:italic>k</jats:italic> <jats:sub>cat</jats:sub> value, retaining less than 1% of the wild‐type enzyme activity. The <jats:italic>k</jats:italic> <jats:sub>cat</jats:sub> values of V33A and K35A were changed slightly, but the <jats:italic>K</jats:italic> <jats:sub>m</jats:sub> of K35A for α‐ketoglutarate was increased to 35.6 mM, compared to the <jats:italic>K</jats:italic> <jats:sub>m</jats:sub> value of 2.5 mM for the wild‐type enzyme. These results suggested that the positive charge at Lys<jats:sup>35</jats:sup> interacted electrostatically with the negative charge at the side chain of α‐ketoglutarate. Site‐directed mutagenesis of the Glu<jats:sup>32</jats:sup> residue was conducted to demonstrate the role of this residue in detail. From the kinetic and spectral characteristics of the Glu<jats:sup>32</jats:sup>‐substituted enzymes, the Glu<jats:sup>32</jats:sup> residue seemed to interact with the positive charge at the Schiff base formed between the aldehyde group of pyridoxal 5′‐phosphate (PLP) and the ε‐amino group of the Lys<jats:sup>145</jats:sup> residue.</jats:p>

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