GENE ANALYSIS OF AN NADP-LINKED ISOCITRATE DEHYDROGENASE LOCALIZED INPEROXISOMES OF THE N-ALKANE-ASSIMILATING YEAST CANDIDA-TROPICALIS

In n-alkane-utilizing yeast, Candida tropicalis, two NADP-linked isoci trate dehydrogenase (NADP-IDH) isozymes are present, one in mitochondr ia (Mt-NADP-IDH) and the other in peroxisomes (Ps-NADP-IDH). Here we r eport the isolation, sequencing, and expression of the gene encoding P s-NADP-IDH (CrIDP2), distinct from the Mt-NADP-IDH gene (CtIDP1). Base d on the N-terminal amino acid sequence of purified Ps-NADP-IDH, a cDN A fragment specific for Ps-NADP-IDH was obtained by the 5'-RACE method . Using this fragment as a probe, the genomic CtIDP2 gene was isolated . Nucleotide sequence analysis of CtIDP2 disclosed that the region enc oding CtIdp2p had a length of 1233 bp, corresponding to 411 amino acid residues. The deduced N-terminal amino acid sequence matched the resu lts obtained from the purified protein. When this CrIDP2 was expressed in Saccharomyces cerevisiae using the C. tropicalis isocitrate lyase gene promoter (UPR-ICL), high intracellular NADP-IDH activity was obse rved. Comparison of amino acid sequences and phylogenetic wee analysis with NADP-IDH enzymes from all reported eukaryotic sources revealed t hat mammalian mitochondrial NADP-IDHs formed a cluster, as did plant N ADP-IDHs. CtIdp2p and other yeast NADP-IDHs were not included in these clusters and seemed to diverge at an early stage from all other enzym es of higher eukaryotes. Ps-NADP-IDH had no typical C-terminal peroxis omal targeting signal and no processing was demonstrated at the N-term inus. However, we could find a region near the N-terminus of the prote in with high similarity to both the putative N-terminal peroxisomal ta rgeting signal sequence of Fox3p of S. cerevisae and an internal regio n of Pox4p of C. tropicalis. The results of northern blot analysis ind icated that the biosynthesis of CtIdp2p was induced in a medium contai ning alkanes as a carbon source, where profuse proliferation of peroxi somes is observed.