HYPERPHENYLALANINEMIA DUE TO DEFECTS IN TETRAHYDROBIOPTERIN METABOLISM - MOLECULAR CHARACTERIZATION OF MUTATIONS IN 6-PYRUVOYL-TETRAHYDROPTERIN SYNTHASE

A variant type of hyperphenylalaninemia is caused by a deficiency of t etrahydrobiopterin (BH4), the obligatory cofactor for phenylalanine hy droxylase. The most frequent form of this cofactor deficiency is due t o lack of 6-pyruvoyl-tetrahydropterin synthase (PTPS) activity, the se cond enzyme in the biosynthetic pathway for BH4. The human liver cDNA for PTPS was previously isolated, and the recombinant protein was foun d to be active when expressed in Escherichia coli. We now have investi gated two patients for their molecular nature of this autosomal recess ive disorder. Both patients were diagnosed as PTPS deficient, one with the central and one with the peripheral form, on the basis of an elev ated serum phenylalanine concentration concomitant with lowered levels of urinary biopterin and PTPS activity in erythrocytes. Molecular ana lysis was performed on the patients' cultured primary skin fibroblasts . PTPS activities were found in vitro to be reduced to background acti vity. Direct cDNA sequence analysis using reverse transcriptase-PCR te chnology showed for the patient with the central form a homozygous G-t o-A transition at codon 25, causing the replacement of an arginine by glutamine (R25Q). Expression of this mutant allele in E. coli revealed 14% activity when compared with the wild-type enzyme. The patient wit h the peripheral form exhibited compound heterozygosity, having on one allele a C-to-T transition resulting in the substitution of arginine 16 for cysteine (R16C) in the enzyme and having on the second allele a 14-bp deletion (Delta 14bp), leading to a frameshift at lysine 120 an d a premature stop codon (K120-->Stop). Heterologous expression of the enzyme with the single-amino-acid exchange R16C revealed only 7% enzy me activity, whereas expression of the deletion allele Delta 14bp exhi bited no detectable activity. All three mutations, R25Q, R16C, and K12 0-->Stop, affect evolutionarily conserved residues in PTPS, result in reduced enzymatic activity when reconstituted in E. coli and are thus believed to be the molecular cause for the BH4 deficiency. This is the first report describing mutations in PTPS that lead to BH4 deficiency .