Phosphatase Subfamily PFKFB

Phosphatase Classification: Fold HP: Superfamily HP (histidine phosphatase): HP, branch1 family: Subfamily PFKFB

PFKFB stands for PFK-2 (6-phosphofructo-2-kinase)/ FBPase-2 (fructose-2,6-bisphosphatase).

Evolution

The FBPase-2 domain of the enzyme subunit bears sequence, mechanistic and structural similarity to the histidine phosphatase family of enzymes. The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo- 1-kinase), but this proved not to be correct. Molecular modelling of the PFK-2 domain revealed that, instead, it has the same fold as adenylate kinase. This was confirmed by X-ray crystallography. A PFK-2/FBPase-2 sequence in the genome of one prokaryote, the proteobacterium Desulfovibrio desulfuricans, could be the result of horizontal gene transfer from a eukaryote distantly related to all other organisms, possibly a protist. This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with possibly only one of the domains active), indicates that fusion of Fru-2,6-P2 (fructose 2,6-bisphosphate) is found in all mammalian tissues, throughout the animal and plant kingdoms, and in fungi and certain unicellular eukaryotes, but not in bacteria (however, see the section on evolution below) (for earlier reviews see [1– 7]). In most of these organisms, this molecule is a potent positive allosteric effector of PFK-1 (6-phosphofructo-1-kinase) (except in some protists in which it is an allosteric stimulator of pyruvate kinase – see below) and thus stimulates glycolysis. In liver, Fru- 2,6-P2 is an inhibitor of FBPase-1 (fructose-1,6-bisphosphatase), a regulatory enzyme of gluconeogenesis. Glucagon decreases the concentration of hepatic Fru-2,6-P2, thereby relieving the inhibition of FBPase-1 and allowing gluconeogenesis to prevail. Therefore Fru-2,6-P2 plays a unique role in the control of glucose homoeostasis by allowing the liver to switch from glycolysis to gluconeogenesis. In most mammalian tissues, which do not contain FBPase-1, Fru-2,6-P2 acts as a glucose signal to stimulate glycolysis when glucose is available. In heart, insulin and anoxia increase Fru-2,6-P2 concentrations, which contributes to the stimulation of glycolysis under these conditions [8,9]. genes initially coding for separate PFK-2 and FBPase-2 domains might have occurred early in evolution. In the enzyme homodimer, the PFK-2 domains come together in a head-to-head like fashion, whereas the FBPase-2 domains can function as monomers. There are four PFK-2/FBPase-2 isoenzymes in mammals, each coded by a different gene that expresses several isoforms of each iso- enzyme. In these genes, regulatory sequences have been identified which account for their long-term control by hormones and tissue- specific transcription factors. One of these, HNF-6 (hepatocyte nuclear factor-6), was discovered in this way. As to short-term control, the liver isoenzyme is phosphorylated at the N-terminus, adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein kinase), leading to PFK-2 inactivation and FBPase-2 activation. In contrast, the heart isoenzyme is phosphorylated at the C-terminus by several protein kinases in different signalling pathways, re- sulting in PFK-2 activation.

Fructose 2,6-bisphosphate is a potent metabolic regulator in eukaryotic organisms; it affects the activity of key enzymes of the glycolytic and gluconeogenic pathways. The enzymes responsible for its synthesis and hydrolysis, 6-phosphofructo-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) are present in representa- tives of all major eukaryotic taxa. Results from a bioinformatics analysis of genome databases suggest that very early in evolution, in a common ancestor of all extant eukaryotes, distinct genes encoding PFK-2 and FBPase-2, or related enzymes with broader substrate specificity, fused resulting in a bifunctional enzyme both domains of which had, or later acquired, specificity for fructose 2,6-bispho- sphate. Subsequently, in different phylogenetic lineages duplications of the gene of the bifunctional enzyme occurred, allowing the development of distinct isoenzymes for expression in different tissues, at specific developmental stages or under different nutritional conditions. Independently in different lineages of many unicellular eukaryotes one of the domains of the different PFK-2/FBPase-2 isoforms has undergone substitutions of critical catalytic residues, or deletions rendering some enzymes monofunctional. In a considerable number of other unicellular eukaryotes, mainly parasitic organisms, the enzyme seems to have been lost altogether. Besides the catalytic core, the PFK-2/FBPase-2 has often N- and C-terminal extensions which show little sequence conservation. The N-terminal extension in particular can vary considerably in length, and seems to have acquired motifs which, in a lineage-specific manner, may be responsible for regulation of catalytic activities, by phosphorylation or ligand binding, or for mediating protein-protein interactions.

Domain

PFKFB has two domains for its two functions [1]:

• The phosphatase FBPase-2 domain is HP2 domain, evidenced by sequence, mechanistic and structural similarity with histidine phosphatases.
• The kinase PFK-2 domain has the same fold with adenylate kinase, confirmed by crystal structure.

Catalytic activity

PFKFB is a homodimeric bifunctional enzyme that catalyses the synthesis and degradation of Fru-2,6-P2 (fructose 2,6-bisphosphate) that is a signal molecule that controls glycolysis [1].