Characterization of glycine substitution mutations within the putative NAD+-binding site of bacillus licheniformis aldehyde dehydrogenase

Yen Chung Lee, Den Tai Lin, Hsiang Ling Chen, Huei Fen Lo, Hui Yu Hu, Nai-Wan Hsiao, Long Liu Lin

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The NAD+-requiring enzymes of the aldehyde dehydrogenase (ALDH) family contain a glycine motif, GX1-2GXXG, which is reminiscent of the fingerprint region of the Rossman fold, a conserved structural motif of the classical nicotinamide nucleotide-binding proteins. In this research, the role of three glycine residues situated within the putative NAD+-binding motif (211-GPGSSAG) together with Gly233 and Gly238 of Bacillus licheniformis ALDH (BlALDH) were probed by site-directed mutatgenesis. Fifteen mutant BlALDHs were obtained by substitution of the indicated glycine residues with alanine, glutamate and arginine. Except for the Ala replacement at positions 211, 213, 217 and 238, the remaining mutant enzymes lost the dehydrogenase activity completely. Tryptophan fluorescence and far-UV circular dichroism spectra allowed us to discriminate BlALDH and the inactive mutant enzymes, and unfolding analyses further revealed that they had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlALDH and the functional variants had a comparable Tm value, but the value was reduced by more than 5.1°C in the rest of mutant enzymes. Acrylamide quenching analysis showed that the inactive mutant enzymes had a dynamic quenching constant greater than that of BlALDH. Native BlALDH started to unfold beyond ∼ 0.21 M GdnHCl and reached an unfolded intermediate, [GdnHCl] 0.5, N-U, at 0.92 M equivalent to free energy change (ΔG N-UH2O) of 12.34 kcal/mol for the N → U process, whereas the denaturation midpoints for mutant enzymes were 0.45-1.61 M equivalent to ΔGN-UH2O of 0.31-4.35 kcal/mol. Taken together, these results strongly suggest that the explored glycines are indeed important for the catalytic activity and structural stability of BlALDH.

Original languageEnglish
Pages (from-to)1183-1193
Number of pages11
JournalProtein and Peptide Letters
Volume19
Issue number11
DOIs
Publication statusPublished - 2012 Nov 1

Fingerprint

Aldehyde Dehydrogenase
Bacilli
NAD
Glycine
Substitution reactions
Binding Sites
Mutation
Guanidine
Enzymes
Denaturation
arginine glutamate
Quenching
Niacinamide
Acrylamide
Dermatoglyphics
Dichroism
Circular Dichroism
Tryptophan
Alanine
Free energy

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Biochemistry

Cite this

Lee, Yen Chung ; Lin, Den Tai ; Chen, Hsiang Ling ; Lo, Huei Fen ; Hu, Hui Yu ; Hsiao, Nai-Wan ; Lin, Long Liu. / Characterization of glycine substitution mutations within the putative NAD+-binding site of bacillus licheniformis aldehyde dehydrogenase. In: Protein and Peptide Letters. 2012 ; Vol. 19, No. 11. pp. 1183-1193.
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abstract = "The NAD+-requiring enzymes of the aldehyde dehydrogenase (ALDH) family contain a glycine motif, GX1-2GXXG, which is reminiscent of the fingerprint region of the Rossman fold, a conserved structural motif of the classical nicotinamide nucleotide-binding proteins. In this research, the role of three glycine residues situated within the putative NAD+-binding motif (211-GPGSSAG) together with Gly233 and Gly238 of Bacillus licheniformis ALDH (BlALDH) were probed by site-directed mutatgenesis. Fifteen mutant BlALDHs were obtained by substitution of the indicated glycine residues with alanine, glutamate and arginine. Except for the Ala replacement at positions 211, 213, 217 and 238, the remaining mutant enzymes lost the dehydrogenase activity completely. Tryptophan fluorescence and far-UV circular dichroism spectra allowed us to discriminate BlALDH and the inactive mutant enzymes, and unfolding analyses further revealed that they had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlALDH and the functional variants had a comparable Tm value, but the value was reduced by more than 5.1°C in the rest of mutant enzymes. Acrylamide quenching analysis showed that the inactive mutant enzymes had a dynamic quenching constant greater than that of BlALDH. Native BlALDH started to unfold beyond ∼ 0.21 M GdnHCl and reached an unfolded intermediate, [GdnHCl] 0.5, N-U, at 0.92 M equivalent to free energy change (ΔG N-UH2O) of 12.34 kcal/mol for the N → U process, whereas the denaturation midpoints for mutant enzymes were 0.45-1.61 M equivalent to ΔGN-UH2O of 0.31-4.35 kcal/mol. Taken together, these results strongly suggest that the explored glycines are indeed important for the catalytic activity and structural stability of BlALDH.",
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Characterization of glycine substitution mutations within the putative NAD+-binding site of bacillus licheniformis aldehyde dehydrogenase. / Lee, Yen Chung; Lin, Den Tai; Chen, Hsiang Ling; Lo, Huei Fen; Hu, Hui Yu; Hsiao, Nai-Wan; Lin, Long Liu.

In: Protein and Peptide Letters, Vol. 19, No. 11, 01.11.2012, p. 1183-1193.

Research output: Contribution to journalArticle

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T1 - Characterization of glycine substitution mutations within the putative NAD+-binding site of bacillus licheniformis aldehyde dehydrogenase

AU - Lee, Yen Chung

AU - Lin, Den Tai

AU - Chen, Hsiang Ling

AU - Lo, Huei Fen

AU - Hu, Hui Yu

AU - Hsiao, Nai-Wan

AU - Lin, Long Liu

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N2 - The NAD+-requiring enzymes of the aldehyde dehydrogenase (ALDH) family contain a glycine motif, GX1-2GXXG, which is reminiscent of the fingerprint region of the Rossman fold, a conserved structural motif of the classical nicotinamide nucleotide-binding proteins. In this research, the role of three glycine residues situated within the putative NAD+-binding motif (211-GPGSSAG) together with Gly233 and Gly238 of Bacillus licheniformis ALDH (BlALDH) were probed by site-directed mutatgenesis. Fifteen mutant BlALDHs were obtained by substitution of the indicated glycine residues with alanine, glutamate and arginine. Except for the Ala replacement at positions 211, 213, 217 and 238, the remaining mutant enzymes lost the dehydrogenase activity completely. Tryptophan fluorescence and far-UV circular dichroism spectra allowed us to discriminate BlALDH and the inactive mutant enzymes, and unfolding analyses further revealed that they had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlALDH and the functional variants had a comparable Tm value, but the value was reduced by more than 5.1°C in the rest of mutant enzymes. Acrylamide quenching analysis showed that the inactive mutant enzymes had a dynamic quenching constant greater than that of BlALDH. Native BlALDH started to unfold beyond ∼ 0.21 M GdnHCl and reached an unfolded intermediate, [GdnHCl] 0.5, N-U, at 0.92 M equivalent to free energy change (ΔG N-UH2O) of 12.34 kcal/mol for the N → U process, whereas the denaturation midpoints for mutant enzymes were 0.45-1.61 M equivalent to ΔGN-UH2O of 0.31-4.35 kcal/mol. Taken together, these results strongly suggest that the explored glycines are indeed important for the catalytic activity and structural stability of BlALDH.

AB - The NAD+-requiring enzymes of the aldehyde dehydrogenase (ALDH) family contain a glycine motif, GX1-2GXXG, which is reminiscent of the fingerprint region of the Rossman fold, a conserved structural motif of the classical nicotinamide nucleotide-binding proteins. In this research, the role of three glycine residues situated within the putative NAD+-binding motif (211-GPGSSAG) together with Gly233 and Gly238 of Bacillus licheniformis ALDH (BlALDH) were probed by site-directed mutatgenesis. Fifteen mutant BlALDHs were obtained by substitution of the indicated glycine residues with alanine, glutamate and arginine. Except for the Ala replacement at positions 211, 213, 217 and 238, the remaining mutant enzymes lost the dehydrogenase activity completely. Tryptophan fluorescence and far-UV circular dichroism spectra allowed us to discriminate BlALDH and the inactive mutant enzymes, and unfolding analyses further revealed that they had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlALDH and the functional variants had a comparable Tm value, but the value was reduced by more than 5.1°C in the rest of mutant enzymes. Acrylamide quenching analysis showed that the inactive mutant enzymes had a dynamic quenching constant greater than that of BlALDH. Native BlALDH started to unfold beyond ∼ 0.21 M GdnHCl and reached an unfolded intermediate, [GdnHCl] 0.5, N-U, at 0.92 M equivalent to free energy change (ΔG N-UH2O) of 12.34 kcal/mol for the N → U process, whereas the denaturation midpoints for mutant enzymes were 0.45-1.61 M equivalent to ΔGN-UH2O of 0.31-4.35 kcal/mol. Taken together, these results strongly suggest that the explored glycines are indeed important for the catalytic activity and structural stability of BlALDH.

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