THE MULTIFUNCTIONAL FOLIC-ACID SYNTHESIS FAS GENE OF PNEUMOCYSTIS-CARINII ENCODES DIHYDRONEOPTERIN ALDOLASE, HYDROXYMETHYLDIHYDROPTERIN PYROPHOSPHOKINASE AND DIHYDROPTEROATE SYNTHASE

The nucleotide sequence of a folic acid synthesis (fas) gene from Pneu mocystis carinii contains an open reading frame (ORF) that predicts a protein of 740 amino acids with an M(r) of 83979. A recombinant baculo virus was constructed which directed expression of the predicted Fas74 0 polypeptide in cultured Spodoptera frugiperda (SF9) insect cells. Th e overexpressed 'full-length' protein migrated anomalously in sodium d odecyl sulfate/polyacrylamide gels, with an apparent molecular mass of 71.5 kDa. An abundant 69-kDa species was also recognized by polyclona l sera specific for the Fas protein in immunoblotting analyses. Dihydr oneopterin aldolase, dihydropterin pyrophosphokinase and dihydropteroa te synthase activities were readily detected in SF9 extracts in which the 71.5/69-kDa immunoreactive species were overproduced, demonstratin g that three enzyme functions involved in catalysing three sequential steps of the folate biosynthetic pathway are encoded by a single gene in P. carinii. Importantly, the polyclonal sera recognize a single 69- kDa species in P carinii extracts suggesting that the three activities are indeed properties of a single polypeptide, although the nature of the suggested post-translational modification is unknown. Location of the individual enzyme domains with the Fas polypeptide based upon ami no acid sequence similarity to their bacterial counterparts is discuss ed. Futhermore, expression of various truncated fas gene constructs de monstrates that the complete fas ORF, including the N-terminus of the predicted polypeptide (FasA domain) whose enzyme function is unknown, must be expressed for maximum dihydroneopterin aldolase (FasB domain) and dihydropteroate synthase (FasD domain) activities. This suggests i nteractions between the domains within the larger polypeptide to stabi lize the functions of these two enzymes. The FasC domain, which contai ns 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase activity, is ab le to fold and function independently of the other domains. The requir ement by mammalian cells for preformed folates, and the absence of dih ydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphok inase and dihydropteroate synthase from these tissues opens up the pos sibility of designing higly selective drugs which inhibit these unique targets.