Characterization of a Thermoacidophilic l-Arabinose Isomerase from Alicyclobacillus acidocaldarius: Role of Lys-269 in pH Optimum

다국어 초록 (Multilingual Abstract)

<B>ABSTRACT</B><P>The <I>araA</I> gene encoding l-arabinose isomerase (AI) from the thermoacidophilic bacterium <I>Alicyclobacillus acidocaldarius</I> was cloned, sequenced, and expressed in <I>Escherichia coli</I>. Analysis of the sequence revealed that the open reading frame of the <I>araA</I> gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of <I>A. acidocaldarius</I> AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to <I>Geobacillus stearothermophilus</I> AI (GSAI) and <I>Bacillus halodurans</I> AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65°C under the assay conditions used, and it required divalent cations such as Mn<SUP>2+</SUP>, Co<SUP>2+</SUP>, and Mg<SUP>2+</SUP> for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent <I>Km</I> values of the recombinant AAAI for l-arabinose and d-galactose were 48.0 mM (<I>V</I>max, 35.5 U/mg) and 129 mM (<I>V</I>max, 7.5 U/mg), respectively, at pH 6 and 65°C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from <I>A. acidocaldarius</I> (pH 6), <I>G. stearothermophilus</I> (pH 7), and <I>B. halodurans</I> (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (<I>k</I>cat/<I>Km</I>) of each mutant at different pHs was significantly affected by an increase or decrease in <I>V</I>max. From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.</P>