2 DOMAINS WITH AMINO-ACID-SEQUENCE SIMILARITY ARE REQUIRED FOR DIHYDRONEOPTERIN ALDOLASE FUNCTION IN THE MULTIFUNCTIONAL FOLIC-ACID SYNTHESIS FAS PROTEIN OF PNEUMOCYSTIS-CARINII

The folic acid synthesized gene (fas) of Pneumocystis carinii (Pc) cod es for a multifunctional enzyme (Fas) known to catalyse three consecut ive steps leading to the production of dihydropteroate in the de novo folate synthesis pathway, Previously, we predicted that a domain, desi gnated FasB (amino acids (aa) 161-280), of the 740-aa multifunctional protein contains the first of the three enzyme activities in the pathw ay, namely dihydroneopterin aldolase (DHNA), since it shares 23% aa id entity with the DHNA of Streptococcus pneumoniae (Sp), We now extend t hese findings to show that a second domain, FasA (aa 39-160), whose fu nction was previously unknown, shares 27% sequence identity with the a djacent FasB domain, indicative of functional similarity. FasA is also 18% identical with the DHNA from Sp. Recombinant baculoviruses were c onstructed which directed the production of either FasA, FasB or FasAB polypeptide species in cultured Spodoptera frugiperda (SF9) insect ce lls. No DHNA activity is associated with either fasA or fasB when prod uced as single domains in the insect-baculovirus system. However, DHNA activity was detected in SF9 extracts containing the overproduced Fas AB polypeptide. The results of aa sequence alignments and expression s tudies suggest that FasA and FasB may be two subunits of the DHNA enzy me moiety within the multifunctional Fas protein of Pc. An alternative interpretation of the results is also discussed.