Rat liver dimethylglycine dehydrogenase. Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone.
Dimethylglycine dehydrogenase (Me2GlyDH), an enzyme of choline catabolism specifically expressed in the mammalian liver, was analyzed in rat hepatocytes in culture. This mitochondrial enzyme carries the FAD cofactor covalently attached to the polypeptide chain by its riboflavin 8 alpha position to N pi of histidine [Cook, R., Misono, K.S. & Wagner, C. (1980) J. Biol. Chem. 259, 12475-12480]. Subcellular fractionation of [14C]riboflavin-labelled hepatocytes and immunoprecipitation with Me2GlyDH-specific antiserum identified a [14C]riboflavin-labelled polypeptide of the size of mature Me2GlyDH only in the mitochondrial fraction. Immunoprecipitation of extracts from [35S]Met-labelled hepatocytes revealed a putative precursor protein to the mature Me2GlyDH in the cytoplasmic fraction. These Me2GlyDH polypeptides were not expressed in cells of the rat hepatoma cell line FAO. A Me2GlyDH cDNA clone of apparent full length was isolated from a rat liver cDNA bank constructed in the plasmid vector pcD-X [Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T. & Arai, K. (1987) Methods Enzymol. 154, 3-28]. The nucleotide sequence of the cDNA contains an open reading frame encoding a protein of 96059 Da. This molecular mass agrees well with the migration on SDS/PAGE of the assumed Me2GlyDH precursor immunoprecipitated from the cytoplasm of [35S]Met-labelled cells. Proteolytic cleavage at the putative mitochondrial processing protease-recognition site Arg(-2)-Ala(-1)-Glu(+1) would lead to the formation of a protein of 91391 Da, which is in good agreement with the estimated 90 kDa of mature Me2GlyDH [Wittwer, A.J. & Wagner, C. (1981) J. Biol. Chem. 256, 4102-4108], and a 43-amino-acid leader peptide. The N-terminus of Me2GlyDH contains a conserved amino acid sequence which forms the dinucleotide-binding site in many enzymes with noncovalently bound FAD. Close to the modified histidine there is an amino acid sequence resembling a sequence conserved in thymidylate synthases and shown in these enzymes to be involved in the binding of the pteroyl polyglutamate cofactor.
Lang
H
H
Biochemisches Institut, Universität Freiburg, Federal Republic of Germany.
Polster
M
M
Brandsch
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D004247:DNA / Q000235:genetics*; D050893:Dimethylglycine Dehydrogenase; D004792:Enzyme Precursors / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics*; D011061:Poly A / Q000235:genetics; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger; D051381:Rats; D012256:Riboflavin / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D004792:Enzyme Precursors; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D005182:Flavin-Adenine Dinucleotide; D011061:Poly A; D012313:RNA; D004247:DNA; D010089:Oxidoreductases, N-Demethylating; D050893:Dimethylglycine Dehydrogenase; C494400:Dmgdh protein, rat; D012256:Riboflavin