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

Felipe Merino and Victoria Guixé

*Laboratorio de Bioqímica y Biología Molecular Facultad de Ciencias Universidad de Chile Chile*

### **1. Introduction**

236 Gene Duplication

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Sugars are one of the most common carbon sources used by heterotrophic organisms. Indeed, sugar phosphorylation is thought to be a key step in the cellular metabolism since, just after transport into the cell, these molecules are phosphorylated to trap them for further metabolic processing. There are several known pathways used to produce pyruvate from the incoming sugar (like glucose or galactose) which is accompanied by the synthesis of ATP and the production of reductive power. Amongst them, the Embden-Meyerhof pathway, or glycolysis, seems to be the most commonly used. Some microorganisms can also use the Entner-Doudoroff pathway. Also, although the pentose phosphate pathway is generally associated with nucleotide synthesis and reductive power in the form of NADPH it also can be linked to the flux from glucose to pyruvate as this pathway has fructose-6-phosphate and glyceraldehyde-3-phosphate as intermediates. Some microorganisms, such as *Lactococcus lactis*, use a pathway very similar to glycolysis, but instead of start from glucose they use galactose as main carbon source. In this fashion glucokinases are replaced by galactokinases and phosphosfructokinases by tagatose-6-phosphate kinases (van Rooijen et al., 1991).

Interestingly, all the above mentioned pathways ultimately converge through glyceraldehyde-3-phosphate. In this way, the main difference between them is what happens with the hexoses. Here, one of the most important reactions are the initial phosphorylations, e.g. phosphorylation of glucose, fructose-6-phosphate, galactose, tagatose-6-phosphate.

Early on the 90s it was already recognized that the transfer of the *γ*-phosphate of ATP to several sugars was catalyzed by at least three different non-homologous protein families: the hexokinase family, the ribokinase family, and the galactokinase family (Bork et al., 1993). The hexokinase family contains enzymes with wide specificities including glucokinases, ribulokinases, gluconokinases, xylulokinases, glycerokinases, fructokinases, rhamnokinases, and fucokinases (Bork et al., 1993). The galactokinase family contains enzymes that catalyze the phosphorylation of galactose, mevalonate, P-mevalonate, and homoserine (Bork et al., 1993). The ribokinase family on the other hand is very interesting since its members catalyze the transfer of the terminal phosphate of ATP to sugars like ribose, fructose, sugar containing molecules such as nucleosides, and sugar phosphate molecules like fructose-6-phosphate, fructose-1-phosphate, and tagatose-6-phosphate (Bork et al., 1993). This makes the ribokinase family the group with the broadest specificity amongst the above mentioned. It is clear that while the three groups share some similar substrates and hence are a great example of

intensively studied. One of the most studied organism here is *Methanocaldococcus jannaschii*<sup>2</sup> (Jones et al., 1983) since it is one of the few organisms known to produce methane at extreme temperatures. Besides it, the *Halobacterium* and *Haloferax* genera are used as models for halophilic organisms while organisms from the *Thermococcus* and *Pyrococcus* genera are used as models of hyperthermophilic organisms. Here, by far, the most studied organism is

On the Specialization History of the ADP-Dependent Sugar Kinase Family 239

In these organisms, sugar degradation proceeds either through the Entner-Doudoroff or the Embden-Meyerhof pathway (Verhees et al., 2003). For instance, members of the *Thermoproteus*, *Thermoplasma*, and *Sulfolobus* genera degrade glucose through a modified version of the Entner-Doudoroff pathway where sugars are phosphorylated only at the 2-keto-3-deoxygluconate or glycerate level. While the former version is still able to produce one ATP molecule per glucose the later does not produce any ATP (for a review see Verhees et al. (2003)). On the other hand, up until the early 90s it was thought that some archaea of the *Euryarchaeota* used a modified unphosphorylated version of the Entner-Doudoroff pathway to degrade glucose (Mukund & Adams, 1991) which was called pyroglycolysis. However, in 1994 it was possible to demonstrate that, in fact, the flux to pyruvate proceeds through a highly modified version of the Embden-Meyerhof pathway (Kengen et al., 1994).Here, although all the intermediates are present, only four of the ten textbook enzymes are conserved (Verhees et al., 2003). In this pathway, the redox reactions are carried out by ferredoxin containing enzymes which latter use the electrons to reduce protons (producing hydrogen) to couple the proton motive force to ATP synthesis by means of a membrane bound hydrogenase enzyme (Sapra et al., 2003). Between the oxido-reductases present in these organisms, perhaps the most interesting is the glyceraldehyde-3-phosphate oxido-reductase. This enzyme is responsible for the single-step conversion of glyceraldehyde-3-phosphate to 3-phosphoglycerate in a phosphate independent manner (Mukund & Adams, 1995). Besides redox reactions, one of the most striking modifications seen in this version of the Embden-Meyerhof pathway is that the phosphorylation of glucose and fructose-6-phosphate is carried out by enzymes that use ADP and not ATP or polyphosphates as the phosphoryl donor (Kengen et al., 1994). These ADP-dependent enzymes are, in fact, homologous to each other and they show no sequence identity over the noise level with any of the hitherto known ATP, or polyphosphate dependent kinases (Tuininga et al., 1999). For this reason it

was initially proposed that they belong to a new protein family called PfkC.

Given that these ADP-dependent enzymes were initially discovered in the hyperthermophilic archaeon *P. furiosus* (Kengen et al., 1994), it has been argued in the literature that the main reason for this "ADP-dependence" is the fact that ADP has a higher thermostability than ATP and also that both nucleotides are essentially equivalent since both have a similar standard ΔG of hydrolysis. However, these arguments are highly misleading since, (i) as metabolism is a non-equilibrium process the free energy change upon phosphoryl transfers depends on the concentration of the metabolites, (ii) several ATP-dependent enzymes can be found in hyperthermophilic organisms, (iii) the ADP-dependent enzymes are also present in mesophilic organisms (see below), and (iv) the half life of ATP at high temperatures is higher than some other metabolic intermediates present in the Embden-Meyerhof pathway (Dörr

The adaptive value of the appearance of the ADP-dependent enzymes has been a matter of great debate. As we have argued before (Guixé & Merino, 2009), it is most likely unrelated

<sup>2</sup> This organism was initially named *Methanococcus jannaschii* and was later renamed as *Methanocaldococcus jannaschii* to aknowledge the fact that those organisms from the *Methanococcus*

*Pyrococcus furiosus*.

et al., 2003).

genus are not thermophilic.

convergent evolution the ribokinase family is the only one that contains enzymes able to phosphorylate sugar phosphates.

In particular, glucokinases have been extensively studied since they are in the top on many metabolic pathways, and hence some sort of metabolic hub, and also they are responsible for most of the flux control in glycolysis (Torres et al., 1988). On the other hand, while in normal conditions the phosphofructokinase from rat liver shows almost no control over the glycolytic flux, in starving conditions it becomes almost as important as glucokinase (Torres et al., 1988) which suggests that they become key in gluconeogenic conditions. Moreover, phosphofructokinases are extensively studied because they are highly regulated enzymes. In this light, phosphofructokinases have also been recognized as one of the key enzymes of glycolysis.

From the ribokinase family, one of the most studied enzyme is the phosphofructokinase-2 from *Escherichia coli* which is often referred to as a member of the PfkB subfamily (Cabrera et al., 2010). It is possible to find a second phosphofructokinase, called phosphofructokinase-11, in the genome of *E. coli* which belongs to another family called PfkA. In this family, the most extensively studied members are the phosphofructokinase-1 from *E. coli* and the phosphofructokinase from *Bacillus stearothermophilus* (Evans et al., 1981; Schirmer & Evans, 1990). Initially it was thought that both PfkB and PfkA groups had a common origin (Wu et al., 1991), but now we know that they are two non-homologous families. Interestingly, while not phylogenetically related, both phosphofructokinase-1 and phosphofructokinase-2 from *E. coli* show strong inhibition at high concentrations of their substrate MgATP (Atkinson & Walton, 1965; Kotlarz & Buc, 1981), which suggests that this is a key requirement of this metabolic step. This reinforces the idea that these enzymes are strongly related to the balance between glycolysis and gluconeogenesis.

Indeed, it has been already demonstrated that the substrate inhibition is needed for the avoidance of a futile cycle of phosphorylation/dephosphorylation of fructose-6-phosphate/fructose-1,6-bisP which will ultimately lead to a net hydrolysis of ATP (Torres et al., 1997). Interestingly, some microorganisms present phosphofructokinases (also members of the PfkA family) which use polyphosphates as a source of phosphate and hence they do not appear to be regulated (Peng & Mansour, 1992).
