A novel thermostable sulfite oxidase from Thermus thermophilus: characterization of the enzyme, gene cloning and expression in Escherichia coli.
A novel sulfite oxidase has been identified from Thermus thermophilus AT62. Despite this enzyme showing significant amino-acid sequence homology to several bacterial and eukaryal putative and identified sulfite oxidases, the kinetic analysis, performed following the oxidation of sulfite and with ferricyanide as the electron acceptor, already pointed out major differences from representatives of bacterial and eukaryal sources. Sulfite oxidase from T. thermophilus, purified to homogeneity, is a monomeric enzyme with an apparent molecular mass of 39.1 kDa and is almost exclusively located in the periplasm fraction. The enzyme showed sulfite oxidase activity only when ferricyanide was used as electron acceptor, which is different from most of sulfite-oxidizing enzymes from several sources that use cytochrome c as co-substrate. Spectroscopic studies demonstrated that the purified sulfite oxidase has no cytochrome like domain, normally present in homologous enzymes from eukaryotic and prokaryotic sources, and for this particular feature it is similar to homologous enzyme from Arabidopsis thaliana. The identified gene was PCR amplified on T. thermophilus AT62 genome, expressed in Escherichia coli and the recombinant protein identified and characterized.
Di Salle
Anna
A
Istituto di Biochimica delle Proteine, CNR, Napoli, Italy.
D'Errico
Giovanni
G
La Cara
Francesco
F
Cannio
Raffaele
R
Rossi
Mosè
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003067:Coenzymes / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005292:Ferricyanides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008982:Molybdenum / Q000378:metabolism; D000090006:Molybdenum Cofactors; D010084:Oxidation-Reduction; D019897:Periplasm / Q000201:enzymology; D011487:Protein Conformation; D011621:Pteridines / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D050876:Sulfite Oxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013447:Sulfites / Q000378:metabolism; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D001426:Bacterial Proteins; D003067:Coenzymes; D005292:Ferricyanides; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D011621:Pteridines; D011994:Recombinant Proteins; D050876:Sulfite Oxidase; D013447:Sulfites; C007931:hexacyanoferrate III; C024687:sodium molybdate(VI); C022261:molybdopterin; C025026:sodium sulfite