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 alpha and beta-subunit of the enzyme phosphofructokinase (Pfk) as heterologous probes to isolate fragments of the respective genes from the dimorphic pathogenic fungus Candida albicans. The complete coding sequences were obtained by combining sequences of chromosomal fragments and fragments obtained by inverse polymerase chain reaction (PCR). The CaPFK1 and CaPFK2 comprise open reading frames of 2961 bp and 2838 bp, respectively, encoding Pfk subunits with deduced molecular masses of 109 kDa and 104 kDa. The genes presumably evolved by a duplication event from a prokaryotic type ancestor, followed 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 not only obtained after coexpression of both genes, but also in conjunction with the respective complementary subunits from S. cerevisiae. This indicates the formation of functional hetero-oligomers consisting of C. albicans and S. cerevisiae Pfk subunits. In C. albicans, specific Pfk activity was shown to decrease twofold upon induction of hyphal growth. CaPfk cross-reacts with a polyclonal antiserum raised against ScPfk and displays similar allosteric properties, i.e. inhibition by ATP and activation by AMP and fructose 2,6-bisphosphate.
Lorberg
A
A
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Kirchrath
L
L
Ernst
J F
JF
Heinisch
J J
JJ
D000494:Allosteric Regulation / Q000235:genetics; D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology* / Q000472:pathogenicity; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D020440:Gene Duplication; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D006019:Glycolysis / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000235:genetics*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D005656:Fungal Proteins; D010732:Phosphofructokinase-1