Disease relevance of pykF

• We report the cloning of the pykA and pykF genes from Escherichia coli, which code for the two pyruvate kinase isoenzymes (ATP:pyruvate 2-O-phosphotransferases; EC 2.7.1.40) in this microorganism [1].
• These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused by mutations in human erythrocyte PK gene [2].
• Pyruvate kinase from Bacillus stearothermophilus is an allosteric enzyme activated by AMP or ribose 5-phosphate but not by fructose 1,6-bisphosphate [3].
• cDNA cloning of human R-type pyruvate kinase and identification of a single amino acid substitution (Thr384----Met) affecting enzymatic stability in a pyruvate kinase variant (PK Tokyo) associated with hereditary hemolytic anemia [4].
• Characterization of growth and acid formation in a Bacillus subtilis pyruvate kinase mutant [5].

High impact information on pykF

• From these results, we concluded that enzymatic stability of the variant was affected by the point mutation of the PK-encoding gene [4].
• To confirm that the nucleotide change was responsible for the variant phenotype, we expressed the L-type PK with the single amino acid change in Escherichia coli and characterized the enzyme [4].
• cDNA clones for human R-type pyruvate kinase (PK) were isolated from a human reticulocyte cDNA library, constructed by PCR with a single gene-specific primer [4].
• The full-length cDNA was 2060 base pairs long, and the cDNA encoded 574 amino acids, the same number as that by rat R-type PK [4].
• The variant PK was thermolabile and moved slowly in the polyacrylamide gel buffered in 10 mM Tris.HCl, pH 8.3; these characteristics were fully compatible with data obtained from the patient's PK [4].

Chemical compound and disease context of pykF

• Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli: the relative roles of these enzymes in pyruvate biosynthesis [1].
• The metabolic response to edd gene knockout in Escherichia coli JM101 and PTS- Glc+ was investigated in gluconate and glucose batch cultures and compared with other pyruvate kinase and PTS mutants previously constructed [6].
• Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media [5].
• It is known that pyruvate kinase of E. coli becomes inactive upon prolonged dialysis in the absence of a reducing reagent, such as dithiothreitol and that the inactive enzyme is reactivated if dithiothretiol is added [7].
• Primary structure of three peptides at the catalytic and allosteric sites of the fructose-1,6-bisphosphate-activated pyruvate kinase from Escherichia coli [8].

Biological context of pykF

• The metabolic regulation of Escherichia coli lacking a functional pykF gene was investigated based on gene expressions, enzyme activities, intracellular metabolite concentrations and the metabolic flux distribution obtained based on (13)C-labeling experiments [9].
• The flux through Pta-Ack pathways and the ethanol production were limited by the available acetyl coenzyme A. It was shown that the glycolysis was activated in pykF mutant in microaerobic culture [10].
• The allosteric regulation of pyruvate kinase [2].
• Both phosphofructokinase and pyruvate kinase were expressed in E. coli cells, and the evidence suggesting that both genes constitute an operon is presented [3].
• There was an open reading frame upstream of the pyruvate kinase gene [3].

Anatomical context of pykF

• Membranes from cells harvested at late exponential phase showed NTP-synthesizing activity and the physical presence of Ndk but not of Pk or Pra [11].
• At these low levels of heterologous gene expression, comparison of the distribution of PYK and PYK/LacZ transcripts across polysome gradients revealed no significant effect mediated by their striking disparity in codon usage [12].
• Nevertheless, upon increasing B-galactosidase mRNA levels, via manipulation of plasmid copy number, a distinct decline in ribosome loading was observed for the heterologous PYK/LacZ transcript which was not mirrored by either endogenous PYK transcripts or other yeast mRNAs of high (Ribosomal protein 1) or moderate (Actin) codon bias [12].

Associations of pykF with chemical compounds

• In contrast, due to decreased availability of pyruvate (and acetyl coenzyme A) in the pykF mutant compared with the wild type, low flux ratios were found through lactate and acetate forming pathways [9].
• Moreover, the overall activity of the ED pathway on gluconate resulted in important increments in PTS- Glc+ and PTS- Glc+ pykF mutants [6].
• It was also found for continuous cultivation that the enzyme activities of the oxidative PP and Entner-Doudoroff pathways increased as the dilution rate increased for the pykF- mutant [13].
• Expression of both isoenzymes was generally higher during xylose fermentation but statistically higher in both strains only for pykF encoding the isoenzyme activated by fructose-6-phosphate, a key metabolite connecting pentose metabolism to the EMP pathway [14].
• In the PTS- glucose+ host background, overexpression of tktA caused a further 3.7-fold increase in carbon flow, while inactivation of pykA and pykF caused a 5.8-fold increase [15].

Regulatory relationships of pykF

• It was found that flux through phosphoenol pyruvate carboxylase and malic enzyme were up-regulated in the pykF- mutant as compared with the wild type, and acetate formation was significantly reduced in the mutant [13].

Other interactions of pykF

• This was also ascertained by the degradation of ptsG mRNA, the gene for which was down-regulated in the pykF mutant [9].
• Expression of a pykF-lacZ fusion was studied as a function of the carbon source in wild-type strains and strains lacking or overproducing the FruR protein of Escherichia coli [16].
• The coordination sphere of Mn2+ observed in E. coli PEPC is similar to that of Mn2+ found in the pyruvate kinase structure [17].
• Considering the characteristics of the enzyme kinetics involved, the working hypothesis was formulated that phosphoenolpyruvate synthase activity was increasingly hampered by rising oxaloacetate and 2-oxoglutarate concentrations, while at the same time pyruvate kinase activity arose due to activation by fructose 1,6-diphosphate [18].

Analytical, diagnostic and therapeutic context of pykF

• Molecular cloning and nucleotide sequence of the gene for pyruvate kinase of Bacillus stearothermophilus and the production of the enzyme in Escherichia coli. Evidence that the genes for phosphofructokinase and pyruvate kinase constitute an operon [3].
• The sequenced region was amplified by the polymerase chain reaction and subsequently transcribed and translated using both in vivo and in vitro systems, and the resultant gene product characterized to show that the gene encodes the type I isoform of pyruvate kinase [19].
• After tryptic digestion, the purification of the labelled peptides and the sequence analysis allowed to identify four modified lysyl residues, namely Lys173, Lys175, Lys272, and Lys317 of the known DNA-deduced sequence of PKI [20].

References