Polymerase chain reaction-based cloning of alkyl-dihydroxyacetonephosphate synthase complementary DNA from guinea pig liver.
Peroxisomes are indispensable organelles for ether lipid biosynthesis in mammalian tissues, and the deficiency of these organelles in a number of peroxisomal disorders leads to deficiencies in ether phospholipids. We have previously purified the committed enzyme for ether lipid biosynthesis, i.e. alkyl-dihydroxyacetone-phosphate synthase, to homogeneity. We have now determined the N-terminal amino acid sequence, as well as additional internal sequences obtained after cyanogen bromide cleavage of the enzyme. With primers directed against the N-terminal sequence and against a cyanogen bromide fragment sequence, a 1100-bp cDNA fragment was obtained by conventional polymerase chain reaction using first-strand cDNA from guinea pig liver as a template. The 5' and 3' ends of the cDNA were obtained by rapid amplification of cDNA ends. The open reading frame encodes a protein of 658 amino acids, containing the N-terminal amino acid sequence as well as the cyanogen bromide cleavage fragment sequences. The derived amino acid sequence includes a mature protein 600 amino acids long and a presequence 58 amino acids long. The latter contains a stretch of amino acids known as peroxisomal targeting signal 2. The size of the mRNA was estimated to be around 4200 nucleotides. Recombinant His-tagged alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli was enzymatically active.
de Vet
E C
EC
Department of Biochemistry of Lipids, Centre for Biomembranes and Lipid Enzymology, Utrecht, The Netherlands.
Zomer
A W
AW
Lahaut
G J
GJ
van den Bosch
H
H
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003488:Cyanogen Bromide / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry*; D006168:Guinea Pigs; D008099:Liver / Q000201:enzymology*; D008830:Microbodies / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D014166:Transferases / Q000235:genetics*
D018076:DNA, Complementary; D010446:Peptide Fragments; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C019654:alkylglycerone-phosphate synthase; D003488:Cyanogen Bromide