Cloning and expression of soluble cytochrome c and its role in polyvinyl alcohol degradation by polyvinyl alcohol-utilizing Sphingopyxis sp. strain 113P3.
The gene encoding cytocrome c in the pva operon of Sphingopyxis sp. strain 113P3 was cloned, on the basis of the sequence of the gene for cytochrome c (GenBank accession no. AB190288). The deduced amino acid sequence of the gene showed homologies (37% and 47% identities) with two cytochromes c of different origins. The recombinant cytochrome c tagged with hexahistidines was expressed in the periplasm of Escherichia coli BL21(DE3) harboring pT-GroE, which was in accordance with the localization of cytochrome c in strain 113P3; the protein was purified to homogeneity. The purified recombinant cytochrome c was a monomeric protein with a molecular weight of 16.5 kDa. The oxidized and reduced forms of the protein showed absorption maxima at 409 nm and at 414, 520 and 550 nm, respectively. The recombinant cytochrome c was fully reduced by polyvinyl alcohol (PVA), coupled with a catalytic amount (1/10 molar concentration) of the recombinant PVA dehydrogenase (PVADH) of the same origin, suggesting that the cytochrome c involved in the pva operon is a physiological primary electron acceptor for PVADH and that PVA dehydrogenation is linked with the respiratory chain in Sphingopyxis sp. strain 113P3.
Mamoto
Rie
R
Faculty of Nutrition, Kobe Gakuin University, Kobe, Hyogo 651-2180, Japan.
Hu
Xiaoping
X
Chiue
Hiroko
H
Fujioka
Yoshio
Y
Kawai
Fusako
F
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020561:Alphaproteobacteria / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D011142:Polyvinyl Alcohol / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility
D011994:Recombinant Proteins; D011142:Polyvinyl Alcohol; D045304:Cytochromes c; D000429:Alcohol Oxidoreductases; C048312:polyvinyl-alcohol dehydrogenase (acceptor)