A novel form of 6-phosphofructokinase. Identification and functional relevance of a third type of subunit in Pichia pastoris.
Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of alpha subunits and alpha/beta subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, alpha, beta, and gamma. The sequence of the gamma subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the gamma subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the gamma subunit is not required for enzymatic activity, the gamma subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and gamma subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the gamma subunit. Therefore, we suggest that the gamma subunit contributes to adaptation of P. pastoris to energy resources.
Tanneberger
Katrin
K
Institute of Biochemistry, Molecular Biochemistry, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany.
Kirchberger
Jürgen
J
Bär
Jörg
J
Schellenberger
Wolfgang
W
Rothemund
Sven
S
Kamprad
Manja
M
Otto
Henning
H
Schöneberg
Torsten
T
Edelmann
Anke
A
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D005434:Flow Cytometry; D005635:Fructosediphosphates / Q000737:chemistry; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000502:physiology*; D010843:Pichia / Q000201:enzymology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D005635:Fructosediphosphates; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1