Cloning and functional expression of a mammalian gene for a peroxisomal sarcosine oxidase.
Sarcosine oxidation in mammals occurs via a mitochondrial dehydrogenase closely linked to the electron transport chain. An additional H2O2-producing sarcosine oxidase has now been purified from rabbit kidney. A corresponding cDNA was cloned from rabbit liver and the gene designated sox. This rabbit sox gene encodes a protein of 390 amino acids and a molecular mass of 44 kDa identical to the molecular mass estimated for the purified enzyme. Sequence analysis revealed an N-terminal ADP-betaalphabeta-binding fold, a motif highly conserved in tightly bound flavoproteins, and a C-terminal peroxisomal targeting signal 1. Sarcosine oxidase from rabbit liver exhibits high sequence homology (25-28% identity) to monomeric bacterial sarcosine oxidases. Both purified sarcosine oxidase and a recombinant fusion protein synthesized in Escherichia coli contain a covalently bound flavin, metabolize sarcosine, L-pipecolic acid, and L-proline, and cross-react with antibodies raised against L-pipecolic acid oxidase from monkey liver. Subcellular fractionation demonstrated that sarcosine oxidase is a peroxisomal enzyme in rabbit kidney. Transfection of human fibroblast cell lines and CV-1 cells (monkey kidney epithelial cells) with the sox cDNA resulted in a peroxisomal localization of sarcosine oxidase and revealed that the import into the peroxisomes is mediated by the peroxisomal targeting signal 1 pathway.
Reuber
B E
BE
Institut für Physiologische Chemie, Ruhr-Universität Bochum, 44780 Bochum, Federal Republic of Germany.
Karl
C
C
Reimann
S A
SA
Mihalik
S J
SJ
Dodt
G
G
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005420:Flavoproteins / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008830:Microbodies / Q000201:enzymology*; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000235:genetics*; D010875:Pipecolic Acids / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D011817:Rabbits; D012521:Sarcosine / Q000378:metabolism; D050879:Sarcosine Oxidase; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000737:chemistry
D018076:DNA, Complementary; D005420:Flavoproteins; D010875:Pipecolic Acids; D005182:Flavin-Adenine Dinucleotide; D011392:Proline; D010089:Oxidoreductases, N-Demethylating; C494349:PIPOX protein, human; D050879:Sarcosine Oxidase; C031345:pipecolic acid; D012521:Sarcosine