ALKYL-DIHYDROXYACETONEPHOSPHATE SYNTHASE - FATE IN PEROXISOME BIOGENESIS DISORDERS AND IDENTIFICATION OF THE POINT MUTATION UNDERLYING A SINGLE ENZYME DEFICIENCY

Peroxisomes play an indispensible role in ether lipid biosynthesis as evidenced by the deficiency of ether phospholipids in fibroblasts and tissues from patients suffering from a number of peroxisomal disorders . Alkyl-dihydroxyacetonephosphate synthase, a peroxisomal enzyme playi ng a key role in the biosynthesis of ether phospholipids, contains the peroxisomal targeting signal type 2 in a N-terminal cleavable presequ ence. Using a polyclonal antiserum raised against alkyl-dihydroxyaceto nephosphate synthase, levels of this enzyme were examined in fibroblas t cell lines from patients affected by peroxisomal disorders. Strongly reduced levels were found in fibroblasts of Zellweger syndrome and rh izomelic chondrodysplasia punctata patients, indicating that the enzym e is not stable in the cytoplasm as a result of defective import into peroxisomes, In a neonatal adrenoleukodystrophy patient with an isolat ed import deficiency of proteins carrying the peroxisomal targeting si gnal type 1, the precursor form of alkyl-dihydroxyacetonephosphate syn thase was detected at a level comparable to that of the mature form in control fibroblasts, in line with an intraperoxisomal localization. A patient with an isolated deficiency in alkyl-dihydroxyacetonephosphat e (DHAP) synthase activity had normal levels of this protein. Analysis at the cDNA level revealed a missense mutation leading to a R419H sub stitution in the enzyme of this patient. Expression of a recombinant p rotein carrying this mutation in Escherichia coli yielded an inactive enzyme, whereas a comparable control recombinant enzyme was active, pr oviding further proof that this substitution is responsible for the in activity of the enzyme and the phenotype. In line with this result is the observation that wild-type alkyl-DHAP synthase activity can be ina ctivated by the arginine-modifying agent phenylglyoxal, The enzyme is efficiently protected against this inactivation when the substrate pal mitoyl-DHAP is present at a saturating concentration. The gene encodin g human alkyl-dihydroxyacetonephosphate synthase was mapped on chromos ome 2q31.