SINGLE AMINO-ACID SUBSTITUTIONS DISRUPT TETRAMER FORMATION IN THE DIHYDRONEOPTERIN ALDOLASE ENZYME OF PNEUMOCYSTIS-CARINII

In the opportunistic pathogen Pneumocystis carinii, dihydroneopterin a ldolase function is expressed as the N-terminal portion of the multifu nctional folic acid synthesis protein (Fas). This region encompasses t wo domains, FasA and FasB, which are 27% amino acid identical. FasA an d FasB also share significant amino acid sequence similarity with bact erial dihydroneopterin aldolases. In the present study, this enzyme fu nction has been overproduced as an independent monofunctional activity in Escherichia coli. Recombinant FasAB-Met23 (amino acids 23-290 of t he predicted open reading frame) was purified and shown to contain dih ydroneopterin aldolase activity. The native FasAB-Met23 is a tetramer of the 30-kDa subunit, demonstrating characteristics of an associating -dissociating equilibrium system in which only the multimeric form of the enzyme is active, Multiple sequence alignment of FasA and FasB wit h other dihydroneopterin aldolases highlights only three positions whe re the amino acid is invariable between all the predicted proteins. Th e role of these conserved amino acid residues in enzyme function was i nvestigated using site-directed mutagenesis. Mutant FasAB-Met23 specie s were overproduced and purified to near homogeneity. Three FasA domai n mutants and two FasB domain mutants had little or no detectable dihy droneopterin aldolase activity, implicating both FasA and FasB in the catalytic mechanism. We show that each mutant protein containing an in activating amino acid substitution has lost its ability to form stable tetramers.