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

Abstract

The phosphofructokinase from the non-conventional yeast Yarrowia 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, h 1·1; S 0·5 52 μM), it was inhibited moderately by MgATP (K i 3·5 mM), and it was strongly inhibited by phosphoenolpyruvate (K i 61 μM). Fructose 2,6-bisphosphate did not activate the enzyme, and AMP and ADP were also without effect. The gene YlPFK1 has 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 that Y. lipolytica has only one gene encoding Pfk, and supports the finding that the enzyme consists of identical subunits. Glucose did not interfere with growth of the Ylpfk1 disruptant 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 in Y. lipolytica, different from the role played by the enzyme in Saccharomyces cerevisiae.

Disruption of Yarrowia lipolytica TPS1 Gene Encoding Trehalose-6-P Synthase Does Not Affect Growth in Glucose but Impairs Growth at High Temperature

“…One possibility is that it may serve to control a yet unrecognized function of hexokinase, another one is that it is a consequence of the protein structure shared by most hexokinases and that organisms with a high glycolytic flux have taken advantage of it to control the first irreversible step of glucose metabolism. In Y. lipolytica differences in kinetic and regulatory properties of important glycolytic enzymes like phosphofructokinase [23] or pyruvate kinase [24] indicate that this yeast regulate glycolysis differently from S. cerevisiae .…”

“…Y. lipolytica is also being used as model to study physiological processes like lipid accumulation [21] or peroxisome biogenesis and pexophagy [22]. Differences in kinetic or regulatory properties of important Y. lipolytica enzymes [23], [24], [25] and in transcriptional regulation of some of its genes with respect to those found in S. cerevisiae [26], [27] have been described. Therefore due to the high sensitivity of Y. lipolytica hexokinase to T6P it appeared worthwhile to isolate the TPS1 gene of this yeast and to analyze the effects of its disruption.…”

Moonlighting Proteins in Yeasts

“…The precise mode of action of the Pfk subunit in the process has not yet been unraveled; also, no role in the process has been reported for the ␤ subunit of Pfk. It would be interesting to study the role, if any, of Pfk in peroxisomal degradation in a yeast such as Yarrowia lipolytica, which grows on substrates that induce peroxisome proliferation, such as alkanes or fats, and whose Pfk has a homo-octameric structure (50).…”

3D structure of phosphofructokinase from Pichia pastoris: Localization of the novel γ-subunits

“…Eukaryotic Pfks can assemble into heterologous tetramers and larger homo- and hetero-oligomers, in contrast to the canonical homotetrameric form found in prokaryotes. Yeasts present the largest diversity; homotetramers in Rhodotorula glutinis (Schröter and Kopperschläger, 1996); homo-octamers in Schizosaccharomyces pombe and Yarrowia lipolytica (Reuter, et al, 2000; Flores, et al, 2005); hetero-octamers in Saccharomyces cerevisiae , Kluyveromyces lactis and Candida albicans (Kopperschläger, et al, 1977; Tijane, et al, 1979; Bär, et al, 1997; Lorberg, et al, 1999); and recently it was discovered that Pichia pastoris Pfk forms either heterododecamers or -tetradecamers (Tanneberger, et al, 2007). …”