title: Contribution of conserved Glu255 and Cys289 residues to catalytic activity of recombinant aldehyde dehydrogenase from Bacillus licheniformis.
abstract: Based on the sequence homology, we have modeled the three-dimensional structure of Bacillus licheniformis aldehyde dehydrogenase (BlALDH) and identified two different residues, Glu255 and Cys289, that might be responsible for the catalytic function of the enzyme. The role of these residues was further investigated by site-directed mutagenesis and biophysical analysis. The expressed parental and mutant proteins were purified by nickel-chelate chromatography, and their molecular masses were determined to be approximately 53 kDa by SDS-PAGE. As compared with the parental BlALDH, a dramatic decrease or even complete loss of the dehydrogenase activity was observed for the mutant enzymes. Structural analysis showed that the intrinsic fluorescence and circular dichroism spectra of the mutant proteins were similar to the parental enzyme, but most of the variants exhibited a different sensitivity towards thermal- and guanidine hydrochloride-induced denaturation. These observations indicate that residues Glu255 and Cys289 play an important role in the dehydrogenase activity of BlALDH, and the rigidity of the enzyme has been changed as a consequence of the mutations.
authors: Department of Bioagricultural Science, National Chiayi University, Chiayi City, Taiwan.
authors: Yen-Chung
authors: YC
authors: Lee
authors: Den-Tai
authors: DT
authors: Lin
authors: Ping-Ling
authors: PL
authors: Ong
authors: Hsiang-Ling
authors: HL
authors: Chen
authors: Huei-Fen
authors: HF
authors: Lo
authors: Long-Liu
authors: LL
authors: Lin
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology*; D002384:Catalysis; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D000444:Aldehyde Dehydrogenase; D003545:Cysteine; D005998:Glycine