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Applied and Environmental Microbiology, December 2005, p. 7888-7896, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.7888-7896.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Department of Biotechnology, Yonsei University, Seoul 120-749, Korea,¹ Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104,² Department of Human Genetics, Emory University, Atlanta, Georgia 30322,³ Food Ingredient Division, CJ Foods R&D, CJ Corporation, Seoul 152-050, Korea,⁴ Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003⁵

Received 28 May 2005/ Accepted 15 August 2005

The araA gene encoding L-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA 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 A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans 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 Mn²⁺, Co²⁺, and Mg²⁺ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent K_m values of the recombinant AAAI for L-arabinose and D-galactose were 48.0 mM (V_max, 35.5 U/mg) and 129 mM (V_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 A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (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 (k_cat/K_m) of each mutant at different pHs was significantly affected by an increase or decrease in V_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.

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