Molecular genetics of phosphofructokinase in the yeast <i>Kluyveromyces lactis</i>

Heinisch, Jürgeni; Kirchrath, Lutz; Liesen, Thomas; Vogelsang, Kathrin; Hollenberg, Cornelis P.

doi:10.1111/j.1365-2958.1993.tb01600.x

Cited by 40 publications

(39 citation statements)

References 54 publications

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“…It has been speculated that the N-terminal half of rabbit muscle Pfk retained its catalytical function, whereas in the C-terminal half a former fructose 6-phosphate binding site has evolved to a fructose 2,6-bisphosphate binding domain with allosteric function (20). As highly homologous regions have been conserved from bacterial through yeast and human Pfk (23) it seems feasible to deduce functions of specific amino acids in the eukaryotic enzymes. Straightforward genetic approaches make yeast the ideal organism to study the effect of point mutations at such sites both in vitro and in vivo, i.e.…”

mentioning

confidence: 99%

A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

Heinisch

Boles

Timpel

1996

Journal of Biological Chemistry

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In this work we used in vitro mutagenesis to modify the allosteric properties of the heterooctameric yeast phosphofructokinase. Specifically, we identified two amino acids involved in the binding of the most potent allosteric activator fructose 2,6-bisphosphate. Thus, Ser 724 was replaced by an aspartate and His 859 by a serine in each of the enzyme subunits. Whereas the substitutions had no drastic effects when introduced only in one of the two types of subunits, kinetic parameters were modified when both subunits carried the mutation. Thus, the enzyme with His 859 3 Ser showed an increase in K a for binding of the activator, whereas the one with Ser 724 3 Asp failed to react to the addition of fructose 2,6-bisphosphate, at all. The enzymes still responded to other allosteric activators, such as AMP. Stabilities of the mutant subunits were not significantly altered in vivo, as judged from Western blot analysis. Phenotypically, strains expressing the mutant PFK genes showed a pronounced effect on the level of intermediary metabolites after growth on glucose. Mutants not responding to the activator at all (Ser 724 3 Asp) also displayed higher generation times on glucose medium. This could be suppressed by increasing the gene dosage of the mutant alleles. These results indicate that fructose 2,6-bisphosphate through its activation of phosphofructokinase plays an important role in regulation of the glycolytic flux.

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mentioning

confidence: 99%

A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

Heinisch

Boles

Timpel

1996

Journal of Biological Chemistry

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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: 79%

“…Therefore, BstBI-fragments of genomic DNA from P. pastoris were investigated by Southern blot analysis. Because a very low similarity was described for the N-termini of the subunits of yeast Pfks (Heinisch et al, 1993), a PCR product obtained with primers PPpPFK2 N-term and PPpPFK2 was used as PpPFK2 -specific probe. One signal was found at about 3800 bp (data not shown).…”

Section: Isolation Of Pppfk2mentioning

confidence: 99%

“…Both subunits, named α and β, are 950 A. Edelmann and J. Bär similar in size and encoded by the genes PFK1 (ScPFK1) and PFK2 (ScPFK2), respectively (Kopperschläger et al, 1977;Heinisch et al, 1989). Two non-identical types of subunits of Pfk, which assemble in equal molar range, were also found in other yeasts, such as Kluyveromyces lactis and Candida albicans (Heinisch et al, 1993;Lorberg et al, 1999). From these data and from alignment of the Pfk amino acid sequences, it was suggested that Pfk subunits of all eukaryotic cells are the result of an intragenic gene duplication of the bacterial Pfk 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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“…Unfortunately, no crystal structure of a eukaryotic phosphofructokinase is known, yet. It should be emphasized that, in phosphofructokinase from S. cere isiae, both types of subunit carry N-terminal extensions of about 180 amino acids, which are not present in phosphofructokinase subunits from other eukaryotic organisms, with the exception of those of the closely related yeast Kluy eromyces lactis [21,22]. No significant similarities could be detected between the first 180 amino acids of either subunit.…”

Section: Introductionmentioning

confidence: 99%

A single point mutation leads to an instability of the hetero-octameric structure of yeast phosphofructokinase

Kirchberger

Edelmann

Kopperschläger

et al. 1999

Biochemical Journal

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Yeast phosphofructokinase is an oligomeric enzyme whose detectable activity in vitro depends on its hetero-octameric structure. Here we provide data demonstrating that an alanine residue at positions 874 (for the PFK1-encoded α-subunit) or 868 (for the PFK2-encoded β-subunit) is crucial to achieve this structure. Thus subunits carrying substitutions by either aspartate or lysine of this residue cause a lack of phosphofructokinase activity in vitro and signals of the subunits are poorly detectable in Western blots. Size-exclusion HPLC in conjunction with ELISA detection of the enzyme protein confirmed that no functional octamer is produced in such mutants. Our data suggest that the mutant subunits, not being assembled, tend to aggregate and subsequently become degraded. Substitution of the alanine by valine in either subunit leads to a reduction in specific activities, as expected from a conservative exchange. The kinetic data of the latter mutant revealed a higher affinity to the substrate fructose 6-phosphate, a lower extent of ATP inhibition and a lower degree of activation by fructose 2,6-bisphosphate. In addition, the affinity of mutants carrying a valine instead of an alanine in either the α- or the β-subunit to fructose 2,6-bisphosphate was increased. As no X-ray data on eukaryotic phosphofructokinases are available yet, our data provide the first evidence that a non-charge amino acid at position 874 or 868 is essential for the formation of the functional oligomer. This conclusion is substantiated by comparison with the structure of the well-known prokaryotic enzyme.

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Molecular genetics of phosphofructokinase in the yeast Kluyveromyces lactis

Cited by 40 publications

References 54 publications

A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

Molecular genetics of 6‐phosphofructokinase in Pichia pastoris

A single point mutation leads to an instability of the hetero-octameric structure of yeast phosphofructokinase

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