Purification, molecular and kinetic characterization of phosphofructokinase‐1 from the yeast Schizosaccharomyces pombe: evidence for an unusual subunit composition

Reuter, Renate; Naumann, Manfred; Bär, Jörg; Haferburg, D.; Kopperschläger, Gèrhard

doi:10.1002/1097-0061(200010)16:14<1273::aid-yea621>3.3.co;2-r

Cited by 8 publications

(19 citation statements)

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“…On this basis, a hetero-oligomeric structure of the enzyme is obvious. These results could not be expected, because homo-as well as hetero-oligomeric Pfks have been found in different kinds of yeasts (Heinisch et al, 1989(Heinisch et al, , 1993Schröter and Kopperschläger, 1996;Lorberg et al, 1999;Reuter et al, 2000). From the evolutionary point of view, homo-oligomeric yeast Pfks appear to be more similar to mammalian Pfk in respect of molecular and kinetic properties as described in the literature.…”

Section: Enzyme Structurementioning

confidence: 78%

“…Pfk from the non-fermentative red yeast Rhodotorula glutinis is a homo-tetrameric enzyme, which shares some kinetic properties with mammalian Pfk (Schröter and Kopperschläger, 1996). Studies on Pfk from the fission yeast Schizosaccharomyces pombe revealed a homo-octameric structure of the enzyme (Reuter et al, 2000). Looking at several other yeasts, one gene (PpPFK1 ) was also described for Pfk from Pichia pastoris (PpPfk) encoding the α-subunit.…”

Section: Introductionmentioning

confidence: 99%

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Molecular genetics of 6‐phosphofructokinase in Pichia pastoris

Edelmann

Bär

2002

Yeast

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Previously, studies on glucose-induced microautophagy in the methylotrophic yeast Pichia pastoris provided evidence that the glucose-induced selective autophagy-1-protein is the α-subunit of 6-phosphofructokinase (Pfk), a key enzyme in the glycolytic pathway. In our work, we could clearly demonstrate that two types of subunits of Pfk exist in P. pastoris. Investigating the yeast cell-free extract by Western blot analysis, two distinct signals of Pfk were obtained. In addition, we isolated a DNA sequence containing the complete ORF of PpPFK2 encoding the β-subunit of Pfk from P. pastoris with a deduced molecular mass of 103.7 kDa. On the basis of these results, a hetero-oligomeric structure of Pfk in P. pastoris became obvious. Because the molecular and kinetic properties of a homo-oligomeric yeast Pfk appear to be more similar to those of mammalian Pfk, as described in the literature, our results are of interest for the growing number of studies on P. pastoris as a heterologous production system. Furthermore, the 3 -and 5 -non-coding regions of PpPFK2 were isolated and several putative binding sites for regulatory factors could be identified in the promoter region. The sequence has been deposited in the GenBank data library under Accession No. AF395078.

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Section: Enzyme Structurementioning

confidence: 78%

Section: Introductionmentioning

confidence: 99%

Molecular genetics of 6‐phosphofructokinase in Pichia pastoris

Edelmann

Bär

2002

Yeast

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“…However, the subunit composition of yeast Pfks varies between species: while the enzymes from S. cerevisiae, K. lactis, P. pastoris and C. albicans are heterooctameric proteins (Kopperschläger et al, 1977;Heinisch et al, 1993;Bär et al, 1997;Lorberg et al, 1999;Kirchberger et al, 2002), those of the fission yeasts Sch. pombe and Y. lipolytica are homo-octameric (Reuter et al, 2000;this work). Thus, the acquisition of an octameric structure does not necessarily result from the interaction between two different types of subunits.…”

Section: Discussionmentioning

confidence: 99%

“…) is about one order of magnitude lower than those known for Pfks from other yeast species, such as S. cerevisiae (Hofmann & Kopperschläger, 1982), Schizosaccharomyces pombe (Reuter et al, 2000), P. pastoris (Kirchberger et al, 2002) and Kluyveromyces lactis (Bär et al, 1997).…”

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confidence: 81%

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

et al. 2005

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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.

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“…Recently, we have purified and characterized a particular form of Pfk from Schizosaccharomyces pombe that is composed of only one type of subunit, forming a homo-octameric structure, and was found to be stable in cell-free extract (Reuter et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

6‐phosphofructokinase from Pichia pastoris: purification, kinetic and molecular characterization of the enzyme

Kirchberger

Bär

Schellenberger

et al. 2002

Yeast

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Abstract6-Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo-affinity dye-ligand chromatography on Procion Blue H-5R-Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6-phosphofructokinase exhibiting activation by AMP and fructose 2,6-bis(phosphate), inhibition by ATP and cooperativity to fructose 6-phosphate. However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6-phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6-phosphate is significantly lower. Aqueous twophase affinity partitioning with Cibacron Blue F3G-A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae. The structural organisation of the active enzyme seems to be different in comparison with hetero-octameric 6-phosphofructokinases from other yeast species. The enzyme was found to be a hetero-oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS-PAGE), respectively, and a third non-covalently complexed protein component (34 kDa, SDS-PAGE). The latter seems to be necessary for the catalytic activity of the enzyme. Sequencing of the N-terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI-TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 [AQNCNVC(L/I)SVHEAHTM] gave no relevant information about the identity of this protein.

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Purification, molecular and kinetic characterization of phosphofructokinase‐1 from the yeast Schizosaccharomyces pombe: evidence for an unusual subunit composition

Cited by 8 publications

References 0 publications

Molecular genetics of 6‐phosphofructokinase in Pichia pastoris

Molecular genetics of 6‐phosphofructokinase in Pichia pastoris

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

6‐phosphofructokinase from Pichia pastoris: purification, kinetic and molecular characterization of the enzyme

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