THE HYDROXYMETHYLDIHYDROPTERIN PYROPHOSPHOKINASE DOMAIN OF THE MULTIFUNCTIONAL FOLIC-ACID SYNTHESIS FAS PROTEIN OF PNEUMOCYSTIS-CARINII EXPRESSED AS AN INDEPENDENT ENZYME IN ESCHERICHIA-COLI - REFOLDING AND CHARACTERIZATION OF THE RECOMBINANT ENZYME

The folic acid synthesis (Fas) protein of Pneumocystis carinii is a mu ltifunctional enzyme containing dihydroneopterin aldolase, 6-hydroxyme thyl-7,8-dihydropterin pyrophosphokinase (PPPK), and dihydropteroate s ynthase activities. Isolation of the stretch of fas cDNA shown by amin o acid similarity to the bacterial counterparts to code for PPPK activ ity (fasC domain) is described. FasC was expressed to high levels in E scherichia coli inclusion bodies using an inducible tac promoter expre ssion system. Solubilization of the inclusion bodies in 6 M guanidine hydrochloride and refolding of the recombinant protein yielded enzymat ically active PPPK which was purified to homogeneity by anion-exchange and gel-filtration chromatography. Sequence analysis showed that the first 13 amino acids of the purified protein were in agreement with th ose predicted from the DNA sequence and, furthermore, that the amino-t erminal methionine had been removed. The enzyme is active in the monom eric form, exhibiting maximum activity at around pH 8.0. Isoelectric f ocusing gave a pI of 9.1. The K(m) value for 6-hydroxymethyl-7,8-dihyd ropterin was 3.6 mum in 50 mM Tris buffer, pH 8.2. The production of i ndependently folded, active P. carinii PPPK will allow detailed bioche mical and structural studies, increasing our understanding of this enz yme domain. (C) 1994 Academic Press, Inc.