**8. References**

16 Will-be-set-by-IN-TECH

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

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

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

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

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.

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

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**1. Introduction** 

process of "recruitment".

activators in venoms of Australian elapid snakes.

**14** 

*1Singapore 2USA* 

**Duplication of Coagulation Factor** 

**Genes and Evolution of Snake** 

**Venom Prothrombin Activators** 

*2Department of Biochemistry, Medical College of Virginia Virginia Commonwealth University, Richmond,Virginia* 

Snake venom is a complex mixture of pharmacologically active molecules which are responsible for immobilization, paralysis, death and digestion of prey organisms. This armory of toxins has evolved to target two key systems, namely the neuromuscular and circulatory systems, in order to induce rapid immobilization and death. So far, several hundreds of protein toxins from snake venoms have been purified and characterized. Most of these toxins have been documented to be structurally, and at times functionally, similar to proteins expressed in different tissues of the body. For example, elapid phospholipase A2 toxins are structurally and catalytically similar to mammalian pancreatic phospholipase A2 enzymes (Robin Doley et al. 2009). Similarly, sarafotoxins are structurally and functionally similar to endothelins produced primarily in endothelium (Landan et al. 1991a). Based on such structural and functional similarities, it is hypothesized that toxin proteins are "recruited" from body proteins by gene duplication (Fry 2005). Accordingly, the genes of body proteins are duplicated and modified to have differential and specific expression in venom glands. This phenomenon is broadly termed as "recruitment". This "recruitment" process of body proteins has not only been observed in snakes but also in various other venomous animals, such as cone snails, spiders, scorpions and sea anemones as well as hematophagous animals (Fry et al. 2009). Although this overarching concept existed in the field of snake venom toxins for decades, there is not much direct molecular evidence for this

Our laboratory has extensively characterized prothrombin activators from Australian elapid snake venoms and documented their structural and functional similarity with mammalian plasma coagulation factors. Through systematic, detailed studies, we provided the molecular details of the "recruitment" of venom prothrombin activators from plasma coagulation factors after gene duplication. We also identified several key structural changes that make these prothrombin activators better toxins. In this chapter, we will describe the first molecular evidence for the "recruitment" process and the evolution of prothrombin

Shiyang Kwong1 and R. Manjunatha Kini1,2 *1Department of Biological Sciences, Faculty of Science* 

*National University of Singapore, Singapore* 

