Cloning, sequencing, and expression of pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii.

Ladror, Uri S.; Gollapudi, Lakshmi Asritha; Tripathi, Rakesh; Latshaw, Steven P.; Kemp, Robert G.

doi:10.1016/s0021-9258(18)55336-9

Cited by 43 publications

(18 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the current study, we have identified a single lysine residue whose modification by PLP brings about the loss of enzyme activity of the PPj-dependent PFK. The position of this residue suggests an alignment of residues between PP¡-PFK and ATP-dependent PFK that differs from that suggested previously (Ladror et al, 1991).…”

contrasting

confidence: 80%

“…Enzymes and Substrates. Pyrophosphate-dependent phosphofructokinase (PPj-PFK) from Propionibacterium freudenreichii was expressed in E. coli bearing the pLGl phagemid (Ladror et al, 1991). Cultures were grown in LB containing 0.5 mM IPTG, and the cells were harvested by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

“…Cultures were grown in LB containing 0.5 mM IPTG, and the cells were harvested by centrifugation. The enzyme was isolated and purified by methods previously described by Ladror et al (1991).…”

Section: Methodsmentioning

confidence: 99%

“…It is suggested that Lys-315 interacts with the 6-phosphate of fructose 6-phosphate and that Lys-80 and -85 may be located near the pyrophosphate binding site. liecently, this laboratory described the amino acid sequence of the pyrophosphate-dependent phosphofructokinase (PR-PFK)1 from Propionibacterium freudenreichii (Ladror et al, 1991). The properties of this enzyme differ substantially from the major family of ATP-dependent phosphofructokinases in that it is dimeric as opposed to being a tetramer and that it displays no allosteric properties.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Identification of critical lysyl residues in the pyrophosphate-dependent phosphofructo-1-kinase of Propionibacterium freudenreichii

Green¹,

Latshaw²,

Ladror³

et al. 1992

Biochemistry

View full text Add to dashboard Cite

Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii was inactivated by low concentrations of the lysine-specific reagent pyridoxal phosphate (PLP) after sodium borohydride reduction. The substrates fructose 6-phosphate and fructose 1,6-bisphosphate protected against inactivation whereas inorganic pyrophosphate had little effect. An HPLC profile of a tryptic digest of PPi-PFK modified at low concentrations of PLP showed a single major peak with only a small number of minor peaks. The major peak peptide was isolated and sequenced to obtain IGAGXTMVQK, where X represents a modified lysine residue, corresponding to Lys-315. Lys-315 was protected from reaction with PLP by fructose 1,6-bisphosphate. As indicated by HPLC maps of PPi-PFK modified with varying concentrations of PLP, a direct correlation was observed between activity loss and the modification of Lys-315. Two of the minor peptide peaks were shown to contain Lys-80 and Lys-85, which were modified in a mutually exclusive manner. Partial protection against modification of these two residues was provided by MgPPi. The data were used to adjust the sequence alignment of the Propionibacterium enzyme with that of ATP-dependent PFK of Escherichia coli to identify homologous residues in the substrate binding site. It is suggested that Lys-315 interacts with the 6-phosphate of fructose 6-phosphate and that Lys-80 and -85 may be located near the pyrophosphate binding site.

show abstract

contrasting

confidence: 80%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Identification of critical lysyl residues in the pyrophosphate-dependent phosphofructo-1-kinase of Propionibacterium freudenreichii

Green¹,

Latshaw²,

Ladror³

et al. 1992

Biochemistry

View full text Add to dashboard Cite

show abstract

“…PPi-PFK was first discovered in Entamoeba histolytica, a parasitic amoeba that infects humans [15]. Subsequently, it was found in diverse organisms, including archaea, bacteria, photosynthetic protists, higher plants and parasitic protozoa such as Giardia, Trichomonas and Toxoplasma [12,[16][17][18][19][20][21]. Although the enzymatic activity and kinetics of a number of PPi-PFKs have been well studied, the physiological roles of PPi-PFKs are much less clear.…”

Section: Introductionmentioning

confidence: 99%

Essential role of pyrophosphate homeostasis mediated by the pyrophosphate-dependent phosphofructokinase in Toxoplasma gondii

et al. 2022

View full text Add to dashboard Cite

Many biosynthetic pathways produce pyrophosphate (PPi) as a by-product, which is cytotoxic if accumulated at high levels. Pyrophosphatases play pivotal roles in PPi detoxification by converting PPi to inorganic phosphate. A number of apicomplexan parasites, including Toxoplasma gondii and Cryptosporidium parvum, express a PPi-dependent phosphofructokinase (PPi-PFK) that consumes PPi to power the phosphorylation of fructose-6-phosphate. However, the physiological roles of PPi-PFKs in these organisms are not known. Here, we report that Toxoplasma expresses both ATP- and PPi-dependent phosphofructokinases in the cytoplasm. Nonetheless, only PPi-PFK was indispensable for parasite growth, whereas the deletion of ATP-PFK did not affect parasite proliferation or virulence. The conditional depletion of PPi-PFK completely arrested parasite growth, but it did not affect the ATP level and only modestly reduced the flux of central carbon metabolism. However, PPi-PFK depletion caused a significant increase in cellular PPi and decreased the rates of nascent protein synthesis. The expression of a cytosolic pyrophosphatase in the PPi-PFK depletion mutant reduced its PPi level and increased the protein synthesis rate, therefore partially rescuing its growth. These results suggest that PPi-PFK has a major role in maintaining pyrophosphate homeostasis in T. gondii. This role may allow PPi-PFK to fine-tune the balance of catabolism and anabolism and maximize the utilization efficiency for carbon nutrients derived from host cells, increasing the success of parasitism. Moreover, PPi-PFK is essential for parasite propagation and virulence in vivo but it is not present in human hosts, making it a potential drug target to combat toxoplasmosis.

show abstract

The Active Site of Pyrophosphate-Dependent Phosphofructo-1-kinase Based on Site-Directed Mutagenesis and Molecular Modeling

Walters

et al. 1998

Archives of Biochemistry and Biophysics

View full text Add to dashboard Cite

Cloning, sequencing, and expression of pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii.

Cited by 43 publications

References 20 publications

Identification of critical lysyl residues in the pyrophosphate-dependent phosphofructo-1-kinase of Propionibacterium freudenreichii

Identification of critical lysyl residues in the pyrophosphate-dependent phosphofructo-1-kinase of Propionibacterium freudenreichii

Essential role of pyrophosphate homeostasis mediated by the pyrophosphate-dependent phosphofructokinase in Toxoplasma gondii

The Active Site of Pyrophosphate-Dependent Phosphofructo-1-kinase Based on Site-Directed Mutagenesis and Molecular Modeling

Contact Info

Product

Resources

About