Gene cloning and biochemical characterization of a NAD(P)+ -dependent aldehyde dehydrogenase from Bacillus licheniformis.
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.
Lo
Huei-Fen
HF
Department of Food Science and Applied Biotechnology, Hungkuang University, 34 Chung-Chie Road, Shalu, Taichung City, Taiwan. hflo@sunrise.hk.edu.tw
Chen
Ya-Jen
YJ
D000444:Aldehyde Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D000447:Aldehydes / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D015870:Gene Expression; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000447:Aldehydes; D009249:NADP; C005556:propionaldehyde; D000444:Aldehyde Dehydrogenase