Genetic and biochemical characterization of phosphofructokinase from the opportunistic pathogenic yeast Candida albicans

We have used the two PFK genes of Saccharomyces cerevisiae encoding the alp ha and beta-subunit of the enzyme phosphofructokinase (Pfk) as heterologous probes to isolate fragments of the respective genes from the dimorphic pat hogenic fungus Candida albicans. The complete coding sequences were obtaine d by combining sequences of chromosomal fragments and fragments obtained by inverse polymerase chain reaction (PCR). The CaPFK1 and CaPFK2 comprise op en reading frames of 2961 bp and 2838 bp, respectively, encoding Pfk subuni ts with deduced molecular masses of 109 kDa and 104 kDa. The genes presumab ly evolved by a duplication event from a prokaryotic type ancestor, followe d by another duplication. Heterologous expression in S. cerevisiae revealed that each gene alone was able to complement the glucose-negative phenotype of a pfk1 pfk2 double mutant. In vitro Pfk activity in S. cerevisiae was n ot only obtained after coexpression of both genes, but also in conjunction with the respective complementary subunits from S. cerevisiae. This indicat es the formation of functional hetero-oligomers consisting of C. albicans a nd S. cerevisiae Pfk subunits. In C. albicans, specific Pfk activity was sh own to decrease twofold upon induction of hyphal growth. CaPfk cross-reacts with a polyclonal antiserum raised against ScPfk and displays similar allo steric properties, i.e. inhibition by ATP and activation by AMP and fructos e 2,6-bisphosphate.