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Volume 272, Number 2, Issue of January 10, 1997 pp. 798-803
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Polymerase Chain Reaction-based Cloning of Alkyl-dihydroxyacetonephosphate Synthase Complementary DNA from Guinea Pig Liver

(Received for publication, August 12, 1996, and in revised form, October 23, 1996)

From the Department of Biochemistry of Lipids, Centre for Biomembranes and Lipid Enzymology, Institute for Biomembranes, Padualaan 8, 3584 CH Utrecht, The Netherlands

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-dihydroxyacetonephosphate 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.

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