**5. Are two ADP-dependent kinases better than one?**

Considering that the glycolysis of *M. jannaschii* is functional with just one enzyme in charge of the phosphorylation of glucose and fructose-6-phosphate it is not clear, at a first glance, why two genes were select by nature in the other members of the *Euryarchaeota*. As it was mentioned above, glucokinases are on the top of several pathways and hence the modification of their activity affects a big part of the metabolism. Indeed, this enzyme generally have a great control of the carbon flux. On the other hand, phosphofructokinases seem to be closely related with the balance between glycolysis and gluconeogenesis. In the archaeon *P. furiosus* it has been shown that the switching between these two metabolic pathways is controlled at the expression level (Schut et al., 2003). When the ADP-dependent phosphofructokinase is expressed the fructose-1,6-bisPase is repressed and *vice versa*. Of course, just shutting the

<sup>5</sup> Glucokinase experiments were performed at 40 °Cgiven the instability of the auxiliar enzyme used.

can produce bifunctional enzymes from specific ones. Strikingly, the sugar discrimination is somehow concentrated in very few hot-spots in the structure. Indeed, the introduction of just one hydrogen bond or some salt bridges seems to modulate the affinity for glucose or fructose-6-phosphate respectively. Unfortunately, to date, we have been unable to absolutely

On the Specialization History of the ADP-Dependent Sugar Kinase Family 253

The gene duplication event itself seems to be related with the separated regulation of the glucokinase and phosphofructokinase activity, along with the balance between glycolysis and gluconeogenesis. Indeed, with two different enzymes a finest tuning of the carbon flux inside

We are very grateful to Dr. Ricardo Cabrera for his help in the creation of this chapter by means of enlightening discussions about the evolutionary implications of the gene duplication in the modified Embden-Meyerhof pathway. This work was supported by Fondo Nacional de

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switch the specificity of these enzymes.

the cell can be achieved.

**7. Acknowledgements**

**8. References**

bifunctional enzyme down in *M. jannaschii* will not only decrease the phosphofructokinase activity, but it will have the undesirable side effect of decreasing the glucokinase activity. In this light, the use of one enzyme for each specificity has a great impact in how the cell can regulate the carbon flux. Indeed, the fact the sugar specificity residues are correlated with some others related with regulation (such as the mutation in the NXXE motif) strongly favors our explanation.

What kind of process produce this? It is now a generally accepted hypothesis that less important genes or parts of a gene tend to change or evolve faster than less important ones, which is known as the Kimura-Ohta principle (for a review see Camps et al. (2007)). It is clear that the upon a gene duplication, the phenotypic effect of a mutation in any copy of these genes should be fairly null with the only exception of those that produces specialized genes. It can be argued that even those mutations that produce inactive proteins which should be deleterious and removed by purifying selection under normal conditions are now nearly neutral since the only extra cost is to produce a non-functional protein.

It has been shown recently that upon a change in the fitness optimum (either produced by an environmental change or an internal redistribution of fluxes) most mutations are fixed by natural selection up until the genes reach a nearly optimal sequence. Then they accumulated mutations according to a neutral model (Razeto-Barry et al., 2011). From the arguments given above, it is clear that the only way in which a duplicated gene can break the neutral regime is when a rare specializing mutation is fixed. In this case, the organism must adapt to the new distribution of internal fluxes. Ohta (1987) reached a similar conclusion based on simulations. He stated: " Positive natural selection favors those chromosomes with more beneficial mutations in redundant copies than others in the population, but accumulation of deteriorating mutations (pseudo genes) have no effect on fitness so long as there remains a functional gene. The results imply the following: Positive natural selection is needed in order to acquire gene families with new functions. Without it, too many pseudo genes accumulate before attaining a functional gene family".

As we have shown here, for our protein family, this would imply just one or two mutations since for instance, just the change of a single interaction can change the balance between both specificities. Of course, upon specialization, mutations that modulate regulation (such as those related with the NXXE motif) increases the flexibility of the metabolism.

Interestingly, although most of the *Euryarchaeota* that present the ADP-dependent kinases have two separated specificities the glucokinase from psychrophilic archaeon *Methanococcoides burtonii* have a big C-terminal deletion that should make it non-functional (Merino & Guixé, 2008). The fact that it is still possible to know that it was a glucokinase suggests that this deletion was recent. The phosphofructokinase gene in this archaeon present a glutamic acid in the position equivalent to E82 in the bifunctional enzyme, which suggests that this could be a bifunctional enzyme too. In this way, it appears that until the phosphofructokinase gene is entirely specialized it still exist the possibility of loosing the specific glucokinase gene.

### **6. Concluding remarks**

Our studies about the evolution of the ADP-dependent sugar kinase family showed that the root of the family is located inside the glucokinase group, demonstrating that the bifunctional (glucokinase/phosphofructokinase) enzyme is not an ancestral form, but could be a transitional form from glucokinase to phosphofructokinase. Unfortunately, to date it has not been possible to obtain the crystal structure of any ADP-dependent phosphofructokinase in the presence of fructose-6-phosphate. However, based on structural modeling we have been able to understand partially the structure/specificity relation up to the point where we can produce bifunctional enzymes from specific ones. Strikingly, the sugar discrimination is somehow concentrated in very few hot-spots in the structure. Indeed, the introduction of just one hydrogen bond or some salt bridges seems to modulate the affinity for glucose or fructose-6-phosphate respectively. Unfortunately, to date, we have been unable to absolutely switch the specificity of these enzymes.

The gene duplication event itself seems to be related with the separated regulation of the glucokinase and phosphofructokinase activity, along with the balance between glycolysis and gluconeogenesis. Indeed, with two different enzymes a finest tuning of the carbon flux inside the cell can be achieved.
