Cloning and functional expression of glucose dehydrogenase complex of Burkholderia cepacia in Escherichia coli.
The thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia sp. SM4 is composed of a catalytic subunit (alpha), an electron transfer subunit (beta), and a small gamma subunit of unknown function. We cloned a 1428-nucleotide gene encoding the beta subunit located immediately downstream of the alpha subunit. This completes the isolation of the genes encoding the three components of the GDH complex, which are clustered very close together with the same transcription polarity in the order gammaalphabeta. The deduced beta subunit amino acid sequence contains three typical heme-binding motifs and was 44-49% identical to the cytochrome c subunits of other FAD-dependent dehydrogenase complexes. The GDHgammaalphabeta complex of B. cepacia was successfully expressed in a fully active form in Escherichia coli by co-expression with cytochrome c maturation genes. Recombinant expression of the GDH complex was also found to restore glucose-dependent respiration in a GDH mutant of E. coli.
Tsuya
Taiki
T
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-13 Naka-machi, Koganei, Tokyo 184-8588, Japan.
Ferri
Stefano
S
Fujikawa
Masako
M
Yamaoka
Hideaki
H
Sode
Koji
K
D000595:Amino Acid Sequence; D016956:Burkholderia cepacia / Q000201:enzymology* / Q000235:genetics*; D019069:Cell Respiration / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D004926:Escherichia coli / Q000502:physiology*; D005947:Glucose / Q000378:metabolism*; D042843:Glucose 1-Dehydrogenase / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D042843:Glucose 1-Dehydrogenase; D005947:Glucose