The dimorphic yeast Yarrowia lipolytica possesses an atypical phosphofructokinase: characterization of the enzyme and its encoding gene.
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; S0.5 52 microM), it was inhibited moderately by MgATP (Ki 3.5 mM), and it was strongly inhibited by phosphoenolpyruvate (Ki 61 microM). 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.
Flores
Carmen-Lisset
CL
Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM and Departamento de Bioquímica, Facultad de Medicina de la Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain.
Martínez-Costa
Oscar H
OH
Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM and Departamento de Bioquímica, Facultad de Medicina de la Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain.
Sánchez
Valentina
V
Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM and Departamento de Bioquímica, Facultad de Medicina de la Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain.
Gancedo
Carlos
C
Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM and Departamento de Bioquímica, Facultad de Medicina de la Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain.
Aragón
Juan J
JJ
Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM and Departamento de Bioquímica, Facultad de Medicina de la Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain.
D000595:Amino Acid Sequence; D003470:Culture Media; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D025221:Phosphofructokinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D003470:Culture Media; D025221:Phosphofructokinases; D005947:Glucose