Distinct metal dependence for catalytic and structural functions in the l-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus

Dong Woo Lee, Eun Ah Choe, Seong Bo Kim, Soo Hyun Eom, Young Ho Hong, Sang Jae Lee, Han Seung Lee, Dong Yun Lee, Yu Ryang Pyun

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Abstract

l-Arabinose isomerase (AI) catalyzes the isomerization of l-arabinose to l-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50°C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K m values are 36 mM for l-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k cat/K m) of the enzyme were 51.4 mM -1 min -1 (l-arabinose) and 0.4 mM -1 min -1 (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70°C, and is strongly activated by Mn 2+. It also has a much higher catalytic efficiency of 4.3 mM -1 min -1 for d-galactose and 32.5 mM -1 min -1for l-arabinose, with apparent K m values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T m = 65-67°C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T m (72°C) than the holoenzyme (78°C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.

Original languageEnglish
Pages (from-to)333-343
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume434
Issue number2
DOIs
Publication statusPublished - 2005 Feb 15

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Geobacillus stearothermophilus
Isomerases
Arabinose
Bacilli
Bacillus
Metals
Galactose
Metal ions
Temperature
Ions
Enzymes
Circular Dichroism
Circular dichroism spectroscopy
Protein folding
Holoenzymes
Denaturation
Gene encoding
Protein Folding
Dichroism
Molecular mass

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Lee, Dong Woo ; Choe, Eun Ah ; Kim, Seong Bo ; Eom, Soo Hyun ; Hong, Young Ho ; Lee, Sang Jae ; Lee, Han Seung ; Lee, Dong Yun ; Pyun, Yu Ryang. / Distinct metal dependence for catalytic and structural functions in the l-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus. In: Archives of Biochemistry and Biophysics. 2005 ; Vol. 434, No. 2. pp. 333-343.
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abstract = "l-Arabinose isomerase (AI) catalyzes the isomerization of l-arabinose to l-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67{\%}). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50°C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K m values are 36 mM for l-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k cat/K m) of the enzyme were 51.4 mM -1 min -1 (l-arabinose) and 0.4 mM -1 min -1 (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70°C, and is strongly activated by Mn 2+. It also has a much higher catalytic efficiency of 4.3 mM -1 min -1 for d-galactose and 32.5 mM -1 min -1for l-arabinose, with apparent K m values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T m = 65-67°C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T m (72°C) than the holoenzyme (78°C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.",
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Distinct metal dependence for catalytic and structural functions in the l-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus. / Lee, Dong Woo; Choe, Eun Ah; Kim, Seong Bo; Eom, Soo Hyun; Hong, Young Ho; Lee, Sang Jae; Lee, Han Seung; Lee, Dong Yun; Pyun, Yu Ryang.

In: Archives of Biochemistry and Biophysics, Vol. 434, No. 2, 15.02.2005, p. 333-343.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Distinct metal dependence for catalytic and structural functions in the l-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus

AU - Lee, Dong Woo

AU - Choe, Eun Ah

AU - Kim, Seong Bo

AU - Eom, Soo Hyun

AU - Hong, Young Ho

AU - Lee, Sang Jae

AU - Lee, Han Seung

AU - Lee, Dong Yun

AU - Pyun, Yu Ryang

PY - 2005/2/15

Y1 - 2005/2/15

N2 - l-Arabinose isomerase (AI) catalyzes the isomerization of l-arabinose to l-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50°C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K m values are 36 mM for l-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k cat/K m) of the enzyme were 51.4 mM -1 min -1 (l-arabinose) and 0.4 mM -1 min -1 (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70°C, and is strongly activated by Mn 2+. It also has a much higher catalytic efficiency of 4.3 mM -1 min -1 for d-galactose and 32.5 mM -1 min -1for l-arabinose, with apparent K m values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T m = 65-67°C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T m (72°C) than the holoenzyme (78°C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.

AB - l-Arabinose isomerase (AI) catalyzes the isomerization of l-arabinose to l-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50°C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K m values are 36 mM for l-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k cat/K m) of the enzyme were 51.4 mM -1 min -1 (l-arabinose) and 0.4 mM -1 min -1 (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70°C, and is strongly activated by Mn 2+. It also has a much higher catalytic efficiency of 4.3 mM -1 min -1 for d-galactose and 32.5 mM -1 min -1for l-arabinose, with apparent K m values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T m = 65-67°C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T m (72°C) than the holoenzyme (78°C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.

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