TYPE II HYPERPROLINEMIA: CLONING AND EXPRESSION IN YEAST OF cDNAs ENCODING HUMAN Δ¹-PYRROLINE-5-CARBOXYLATE (P5C) DEHYDROGENASE AND DELINEATION OF MUTATIONS RESPONSIBLE FOR TYPE II HYPERPROLINEMIA (HP II).• 860

P5C dehydrogenase (P5CDh; EC 1.5.1.12) is a mitochondrial matrix NAD⁺ dependent enzyme catalyzing the conversion of P5C, derived either from proline or ornithine, to glutamate. This reaction is a component in the pathway interconnecting the urea and tricarboxylic acid cycles and is deficient in HPII, an autosomal recessive disorder characterized by seizures and accumulation of proline and P5C. To understand the normal biochemistry of P5CDh and the molecular basis of HPII, we utilized peptide sequence data and degenerate primer PCR to clone two full length human P5CDh cDNAs, differing in length by 1 kb (pH sP5CDhS and pHsP5CDhL). Both have a 1689 bp ORF encoding a protein of 563 residues with a predicted molecular mass of 60 kDa. P5CDhL is the predominant transcript and contains an additional 1 kb insert in the 3′ untranslated region that appears to be an alternatively spliced intron. To confirm the identity of the putative human P5CDh cDNAs, we transfected them into a P5CDh-deficient S. cerevisiae strain (MB1472, [put2]). Both restored the ability of these cells to grow with proline as a sole nitrogen source and yielded detectable P5CDh activity (Table). Using RT/PCR we amplified P5CDh cDNAs from four unrelated HPII patients and found mutations in all including one proband from the large Irish Travelers pedigree. Currently we are testing the functional consequence of these mutations using the yeast expression system.

Table 1