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Genetic and biochemical characterization of phosphofructokinase from the opportunistic pathogenic yeast Candida
A Lorberg1, L Kirchrath, J F Ernst
1Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Abstract:
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.
Insights
Researchers isolated and characterized two phosphofructokinase (PFK) genes from Candida albicans. These genes enable functional PFK enzyme production in yeast, revealing insights into fungal metabolism and evolution.
Area of Science:
- * Molecular biology
- * Mycology
- * Biochemistry
Background:
- * Phosphofructokinase (PFK) is a key enzyme in glycolysis.
- * Understanding PFK in pathogenic fungi like Candida albicans is crucial for metabolic studies.
Purpose of the Study:
- * To isolate and characterize the PFK genes (CaPFK1 and CaPFK2) from Candida albicans.
- * To investigate the functional expression and properties of these genes.
Main Methods:
- * Used Saccharomyces cerevisiae PFK genes as probes for gene isolation.
- * Employed inverse polymerase chain reaction (PCR) to obtain complete coding sequences.
- * Performed heterologous expression in S. cerevisiae and in vitro enzyme activity assays.
Main Results:
- * Isolated complete coding sequences for CaPFK1 (2961 bp) and CaPFK2 (2838 bp).
- * Both genes complemented glucose metabolism defects in S. cerevisiae.
- * Demonstrated formation of functional hetero-oligomers between C. albicans and S. cerevisiae PFK subunits.
- * Observed a twofold decrease in specific PFK activity in C. albicans during hyphal growth.
- * Characterized CaPfk allosteric properties, including inhibition by ATP and activation by AMP and fructose 2,6-bisphosphate.
Conclusions:
- * The two PFK genes in C. albicans likely evolved from a common ancestor.
- * Functional PFK enzymes can be formed by combining subunits from C. albicans and S. cerevisiae.
- * PFK activity is regulated by hyphal growth in C. albicans, suggesting a role in dimorphism.