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- W1965121010 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/sub> 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 (Δ G H2ON-U)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 Δ G H2ON-U 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. Keywords: Aldehyde dehydrogenase, NAD+-binding motif, site-directed mutagenesis, thermal unfolding, guanidine hydrochloride" @default.
- W1965121010 created "2016-06-24" @default.
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- W1965121010 date "2012-09-01" @default.
- W1965121010 modified "2023-10-05" @default.
- W1965121010 title "Characterization of Glycine Substitution Mutations within the Putative NAD+-binding Site of Bacillus licheniformis Aldehyde Dehydrogenase" @default.
- W1965121010 doi "https://doi.org/10.2174/092986612803217097" @default.
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