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

Abstract

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 Mn 2؉ , Co 2؉ , and Mg 2؉ 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.

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

“…Nevertheless, the majority of L-AIs have high optimum pHs, which is a major drawback for industrial applications, since isomerization at high temperatures and under alkaline conditions leads to unwanted side reactions generating undesirable subproducts (19). Recently, the key role of K269 in the acidotolerance of Alicyclobacillus acidocaldarius L-AI was reported (19).…”

“…Recently, the key role of K269 in the acidotolerance of Alicyclobacillus acidocaldarius L-AI was reported (19). A mutation introduced at the equivalent position (D268K) in Bacillus halodurans L-AI decreased the optimum pH of the enzyme from 8.0 to 7.0 (19).…”

Strain improvement studies on <i>Microbacterium foliorum</i> GA2 for production of α-amylase in solid state fermentation: Biochemical characteristics and wash performance analysis at low temperatures

“…This unexplored microbial diversity represents an untapped source of potentially novel and unique enzymatic activities and metabolic pathways. Unfortunately, for industrial purposes, high temperatures and an alkaline pH result in the formation of side reactions generating undesirable by-products (together with metal ions) that need to be removed at the end of the process, thereby increasing the production costs (Lee et al, 2005;Rhimi et al, 2007). Microorganisms growing in low-temperature regions are important for their enzymes with potential industrial applications.…”

“…Such cold-adapted microorganisms offer a wide biotechnological potential over the use of organisms and their enzymes that operate at higher temperatures (Kuddus and Ramteke, 2009). Protein engineering has also been used to generate enzymes acting at low temperatures (Lee et al, 2005;Prabhu et al, 2010;Rhimi et al, 2009). Mutation induction and/or selection techniques, along with cloning and protein engineering strategies, have been used extensively to develop enzymes with a higher activity (Kitamura and Kamei, 2003).…”

Homologous Alkalophilic and Acidophilic l -Arabinose Isomerases Reveal Region-Specific Contributions to the pH Dependence of Activity and Stability

“…TP7 AI [ASAI]). The presumed ASAI amino acid sequence showed high (68% to 96%) similarity to those of other AIs from A. acidocaldarius (7,19,21) and Geobacillus stearothermophilus (17).The characterization of purified ASAI is summarized in Table S1 in the supplemental material. The relative molecular weight (M r ) of ASAI was estimated to be 56 based on SDS-PAGE.…”