- ID: MMSYN1_0220
- Name: pfk (ATP-dependent 6-phosphofructokinase)
- Organism: JCVI-Syn3.0
- UniProt ID: http://www.uniprot.org/uniprot/F4MPH4
The gene codes for the ATP-dependent 6-phosphofructokinase enzyme. 6-phosphofructokinase catalyzes the first step of glycolysis, that is, the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate by ATP:
ATP + D-fructose 6-phosphate = ADP + D-fructose 1,6-bisphosphate
The enzyme uses Mg2+ as a cofactor (http://www.uniprot.org/uniprot/F4MPI1). Its activity is allosterically regulated: ATP and phosphoenolpyruvate inhibit it and ADP activates it, depending on the cell’s needs. 6-phosphofructokinase has shown to have 2 lobes, alpha and beta: one binds ATP and the other is involved in substrate-bindings and contains the allosteric site. It exists as a homotetramer in mammals and bacteria, meaning that each monomer contains two identical domains. The domains have 2 main states - the ‘closed’ state and the ‘open state’. In the ‘closed’ state, the magnesium ion brings the two products of the reaction closer together. In the ‘open state’, only ADP binds to the magnesium, which drives the products further apart from each other. These stages are thought to be the successive stages of the reaction (https://en.wikipedia.org/wiki/Phosphofructokinase).
- DNA Length: 981 base pairs.
- DNA sequence:
ATG ATT AAG AAG ATA GGA ATA TTA ACC TCT GGA GGA GAT GCT CCG GGC ATG AAT AAT GCT ATT GCT GGT GTG GTT AAG ACC GCG GCG TCC AAG GGA ATC GAA GTG TAC GGT ATC AAC GAC GGT TAC AAG GGG TTA GTT AAT GGT TGG TTT GAA AAG TTA AAC GTT GAG AAG ACA TTG GAC ATC CTG AGT CGC GGA GGT ACA TAT CTT GGA TCT GCT CGG TTG CCC GAA TTT AAA GAA TTG GAG GTT CGC CAG AAA GCA GTT GCG AAT TTA AAG AAG GCG GGA ATT GAG GCC CTT GTT GTT ATA GGT GGA GAC GGT TCA TAC ATG GGG GCT CAA AAG CTG ACG GAA ATG GGG ATT AAC TGC ATT GGA CTG CCG GGT ACA ATT GAT AAC GAT ATA AGT AGC ACC GAT TAT ACA ATA GGC TTC CAT ACT GCT TTA AAC ACC ATA GTA GAA GCT GTT GAT CGG ATC AGA GAT ACC ATG CAA TCC CAT AAC AGA GCT GAC GTT GTC GAA ATC ATG GGG AAT GGG TGT GGC GAC TTG GTC ACT TAC ACT GCG GTA GCT ACG GGG GCT GAA ATT TTT TCT CCG GCA GAG GAC CTT CTT ACC ATT GAA CAG ATG GGT CAG AAG GCT AAA CAA TTT CGC TTA TTA GGG AAG AAA AGT TTG ATT ATA CTG GTG AGC GAG AAG AGT TAC GGT ATT TCT GCT GAA GAG ATC GCA GAG AAG ATC CAG AAA ATT TCC GGC TAT GAG ACT AAG GCA ACA GTC CTG GGA CAT ATT CAG CGT GGC GGG CGG CCC ACG GCG ATG GAT CGC TAT ATC GCA TTC ACC GCC GGT ATG TTT GCT GTG GAA AAG TTA GCT GAG GGA AAG GGA GGT TTA TTC ATC GGT TTG GAA AAT AAC AAA CTG GTG GCT AGA GAC ATC GAT TCC ACA CTG AAT ATG AAG AAA GAA GAC AAA AAG CCT TTC ATT AAT TAC CTG CGC GAA ATC AAT GGG TAC TTT TCT AAA TAG
- Amino Acid length: 326 amino acids.
- Amino Acid sequence:
Function and Homologs
- Functional Category: Glucose transport & catabolism.
- Product: 6-phosphofructokinase
- Module: glycolitic pathway
- Closest homologous proteins: The top (max three) homologous proteins to this protein, as identified by BLAST searches.
- 6-phosphofructokinase [Mycoplasma leachii], Max score = 656/Query Cover = 100% /E-Value = 0.00 /Ident = 99%, [WP_013447579.1]
- 6-phosphofructokinase [Mycoplasma mycoides subsp. mycoides SC str. Gladysdale], Max score = 654/Query Cover = 100% /E-Value = 0.00 /Ident = 99%, [ADK69606.1]
- 6-phosphofructokinase [Mycoplasma feriruminatoris], Max score = 651/Query Cover = 100% /E-Value = 0.00 /Ident = 98%, [WP_008363698.1]
- Equivalent E. coli / JCVI functional protein: EG10699
- Expression Level: low
- Expression Level Hypothesis: Although 6-phosphofructokinase plays an important role in cell metabolism, it has only 1 function i.e. the catalysis of the first step of glycolysis. Therefore, the cell does not need to produce a lot of 6-phosphofructokinase, and the pfk gene expression level is low. In addition, JCVI probably uses other metabolites instead of 6-phosphofructokinase, so the cell only needs it in small amounts.
- Expression Level References and Description: I used the Mycoplasma genitalium database (File:MgenitaliumSimProteinCounts.xlsx). I am sure about my choice because I found the exact same protein in Mycoplasma genitalium, so their expression levels must be the same.
- Expression Time: early
- Expression Level Hypothesis: 6-phosphofructokinase plays an important role in respiration, which is a vital process that occurs in all cells constantly. However, it would be more important for pfk to be expressed early on in the development of the organism, so that it has a chance to grow.
- Expression Time References and Description: I made this decision based on the protein's function & did not use any resources.
- Other Components: MMSYN1 0729
- Possible Dependencies: enzymes, such as amylase, maltase and sucrase, that break down polysaccharide chains into glucose. Glucose is required for glycolysis to be able to occur.
- Process: glycolysis (the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate)
- Inputs: ATP + D-fructose 6-phosphate
- Outputs: ADP + D-fructose 1,6-bisphosphate
- References: http://www.uniprot.org/uniprot/F4MPH4
- Synthesis Score: The synthesis score of your construct: 1, 2,3
- Predicted Translation Rate: Prediction of construct translation rate from the RBS calculator
- Design Notes and Details: For example, had to use a rare codon to fix folding energy;
- GenBank File: A link to the GenBank file. file