Gene cloning and characterization of dihydrolipoamide dehydrogenase from Microbacterium luteolum: A useful enzymatic regeneration system of NAD+ from NADH.
Dihydrolipoamide dehydrogenase (LPD), a useful biocatalyst for regenerating NAD(+), was purified from Microbacterium luteolum JCM 9174, and the gene encoding LPD was cloned from the genomic DNA. The gene contained an opening reading frame consisting of 1395 nucleotides encoding 465 amino acid residues with a predicted molecular weight of 49912.1 Da, which displayed 36-78% homology to known LPDs. Moreover, the FAD- and NAD(+)-binding sites and the two catalytic residues in the LPDs were conserved. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by column chromatography. LPD of M. luteolum (MluLPD) accepted not only lipoamide but also some artificial electron acceptors such as dichlorophenolindophenol (DCIP) and nitrotetrazolium blue (NTB), that is, it functions as a diaphorase. NAD(+) demonstrated a strong activating effect on MluLPD, and the activity was 5.2 times higher than that without NAD(+). The enzyme was suitable for regenerating NAD(+) in biocatalytic reactions because of its high affinity for NADH (6.1 microM). An NAD(+)-regenerating system with MluLPD and laccase using 2,5-dimethoxy-1,4-benzoquinone as a hydrogen acceptor was demonstrated.
Department of Biotechnology, Faculty of Engineering (Biotechnology Research Center), Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Junji
J
Kurokawa
Manabu
M
Asano
Shunsuke
S
Nomoto
Yoshihide
Y
Makino
Nobuya
N
Itoh
D003001:Cloning, Molecular / Q000379:methods; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008835:Micrococcaceae / Q000201:enzymology* / Q000235:genetics; D009243:NAD / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*
D009243:NAD; D008058:Dihydrolipoamide Dehydrogenase