Gene Cloning and Biochemical Characterization of a NAD(P)+-Dependent Aldehyde Dehydrogenase from Bacillus licheniformis

Abstract

A putative aldehyde dehydrogenase (ALDH) gene, ybcD (gene locus b1467), was identified in the genome sequence of Bacillus licheniformis ATCC 14580. B. licheniformis ALDH (BlALDH) encoded by ybcD consists of 488 amino acid residues with a molecular mass of approximately 52.7 kDa. The coding sequence of ybcD gene was cloned in pQE-31, and functionally expressed in recombinant Escherichia coli M15. BlALDH had a subunit molecular mass of approximately 53 kDa and the molecular mass of the native enzyme was determined to be 220 kDa by FPLC, reflecting that the oilgomeric state of this enzyme is tetrameric. The temperature and pH optima for BlALDH were 37 degrees C and 7.0, respectively. In the presence of either NAD(+) or NADP(+), the enzyme could oxidize a number of aliphatic aldehydes, particularly C3- and C5-aliphatic aldehyde. Steady-state kinetic study revealed that BlALDH had a K (M) value of 0.46 mM and a k (cat) value of 49.38/s when propionaldehyde was used as the substrate. BlALDH did not require metal ions for its enzymatic reaction, whereas the dehydrogenase activity was enhanced by the addition of disulfide reductants, 2-mercaptoethanol and dithiothreitol. Taken together, this study lays a foundation for future structure-function studies with BlALDH, a typical member of NAD(P)(+)-dependent aldehyde dehydrogenases.

Novel NAD+-Farnesal Dehydrogenase from Polygonum minus Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant

“…Concentration for each of these inhibitors and metal ions was set to be 0.1 mM and the incubated enzyme was assayed according to the standard assay method as described before. These inhibitors and metal ions were widely used in many previous reports of aldehyde dehydrogenase [43–48], and thus were selected for this study. The enzyme activity obtained from the reaction mixture without any extra ion or inhibitor was taken as a control, corresponding to 100% relative activity.…”

Isolation and cloning of an aryl-aldehyde dehydrogenase gene from the white-rot fungus Pycnoporus cinnabarinus strain MUCL 39533

“…Other matches of more than 65% identity were to ALDH of Punctularia strigosozonata (GenBank Accession EIN03470.1), NADdependent ALDH of Fomitiporia mediterranea (GenBank Accession EJD04639.1) and ALDH of Coniophora puteana (GeneBank Accession EIW75974.1). Sixteen amino acid residues were fully conserved in more than 95% of the sequences (Perozich et al, 1999;Lo and Chen, 2010;Yao et al, 2012) (Figure 2). PcALDH has two active-site residues Glu268 and Cys296 that are responsible for catalytic activity (Lo and Chen, 2010).…”

“…Sixteen amino acid residues were fully conserved in more than 95% of the sequences (Perozich et al, 1999;Lo and Chen, 2010;Yao et al, 2012) (Figure 2). PcALDH has two active-site residues Glu268 and Cys296 that are responsible for catalytic activity (Lo and Chen, 2010). A glycine motif, presumably involved in NAD(P) + binding, GXGXXXG (GYGHVVG) was found between motifs 3 and 4.…”

“…During acylation, a cysteine nucleophile interacts with the carbonyl carbon of aldehyde forming a thiohemiacetal intermediate, followed by hydride transfer from a tetrahedral thiohemiacetal intermediate to the pyridine ring of NAD(P) + . Then, deacylation occurs involving hydrolysis of the resulting thioester intermediate (Marchal et al, 2001;Lo and Chen, 2010;Munoz-Clares et al, 2011;Tsybovsky et al, 2013). Glu residues play important roles in activation of water molecules (Marchal et al, 2001).…”

Identification and Molecular Characterization of Genes Coding Pharmaceutically Important Enzymes from Halo-Thermo Tolerant Bacillus

“…27 Aldehyde dehydrogenase (ALDH; aldehyde: NAD(P) + oxidoreductase, EC 1.2.1.5) constitute a group of enzymes that catalyze the conversion of aldehydes to the corresponding acids mediated by an NAD(P) + -dependent virtually irreversible reaction making it potentially useful in an industrial settings. The ALDH is very unstable because of the spontaneous oxidation, 34 therefore, there is considerable interest in production of stable ALDH, which can be used more efficiently in the pharmaceutical and fine chemicals industries for the production of aldehydes, ketones, and chiral alcohols. The production of chiral compounds is particularly desired because this is an increasingly important step in the synthesis of chirally pure pharmaceutical agents.…”