The dimorphic yeast Yarrowia lipolytica possesses an atypical phosphofructokinase: characterization of the enzyme and its encoding gene

Abstract

The phosphofructokinase from the non-conventional yeastYarrowia lipolytica(YlPfk) was purified to homogeneity, and its encoding gene isolated. YlPfk is an octamer of 869 kDa composed of a single type of subunit, and shows atypical kinetic characteristics. It did not exhibit cooperative kinetics for fructose 6-phosphate (Hill coefficient,h1·1;S0·552 μM), it was inhibited moderately by MgATP (Ki3·5 mM), and it was strongly inhibited by phosphoenolpyruvate (Ki61 μM). Fructose 2,6-bisphosphate did not activate the enzyme, and AMP and ADP were also without effect. The geneYlPFK1has no introns, and encodes a putative protein of 953 aa, with a molecular mass consistent with the subunit size found after purification. Disruption of the gene abolished growth in glucose and Pfk activity, while reintroduction of the gene restored both properties. This indicates thatY. lipolyticahas only one gene encoding Pfk, and supports the finding that the enzyme consists of identical subunits. Glucose did not interfere with growth of theYlpfk1disruptant in permissive carbon sources. The unusual kinetic characteristics of YlPfk, and the intracellular concentrations of glycolytic intermediates during growth in glucose, suggest that YlPfk may play an important role in the regulation of glucose metabolism inY. lipolytica, different from the role played by the enzyme inSaccharomyces cerevisiae.