**2. Biosynthesis of menaquinone and phylloquinone**

The major quinones found in nature are ubiquinone (UQ), menaquinone (MK), and phyllo‐ quinone (K1) (**Figure 1**). They differ not only in structure but also in their redox potentials, so the incorporation of one or the other as a cofactor allows for fine‐tuning of electron transfer reactions. The distribution in nature of genes involved in menaquinone biosynthesis suggests that it was most likely the original quinone; this is supported by the observation that mena‐ quinone is readily oxidized in aerobic environments, suggesting that it existed long before the appearance of oxygen [6]. There are many different species of menaquinone, though they differ only in the length of their isoprenyl side chains. These differences are reflected in the nomen‐ clature of menaquinones, wherein the number of isoprene units is indicated (i.e., MK‐4). Phylloquinone, usually considered distinct from the menaquinones, is merely MK‐4 with a more heavily saturated lipophilic tail.

**Figure 1.** Structures of common quinones.

ions back out into the surrounding milieu. These early efflux pumps most likely exported protons at the expense of ATP, which the cells were forced to make through substrate‐level phosphor‐ ylation, an inefficient process. However, leakage of protons back into the cell could drive the ATPase in the reverse direction, thus linking the extrusion of protons to the creation of cellular

Aside from the issue of osmotic pressure, the plasma membrane also created a conundrum in that the organic molecules necessary to drive metabolism were prevented entry. Thus, cells developed membrane‐associated transporters capable of importing such nutrients. Catabo‐ lism of these organic molecules provided the cells with necessary building blocks and resulted in the liberation of electrons, which could then be collected on redox‐active molecules like

To regenerate the pools of electron carriers, these ancient microbes resorted to fermentation, the process by which electrons are dropped onto self‐derived organic molecules. This freed up NAD+ and FAD+ to participate in more rounds of catabolism of carbon sources, thus driving metabolism. However, fermentation is an inefficient process and thus limited the growth rate and abundance of these microbes. Only when the enzymes involved in fermentation (i.e., nitrate reductase, fumarate reductase) evolved to associate with the plasma membrane did these ancient microbes begin to tap into the power they needed to flourish. Now, the process of passing electrons onto terminal acceptors could be coupled to the extrusion of protons into the extracellular space. With greater numbers of protons pumped out of the cell, their leakage back across the membrane could greatly increase the amount of ATP generated [1]. In essence, the cells could target these molecular machines to the membrane to produce the chemical energy necessary to fuel metabolism. The effect could be further amplified by linking these redox reactions together, but that required cofactors capable of accepting and donating electrons to act as molecular wires. The earliest such cofactors were likely iron‐sulfur clusters and flavins, but these were not readily inserted into the highly lipophilic environment of the plasma membrane. Thus arose the quinones, which are fat‐soluble redox molecules capable of associating with membrane‐embedded enzymes. By linking together several modular redox complexes into an electron transport chain capable of extruding protons, quinones potentiated a huge leap forward in bioenergetics and greatly increased the capacity for complexity in

To maximize the utility of having quinones available in their membranes, these ancient microbes needed to find a way to tap into more plentiful electron donors and acceptors. One potential source of electrons present in abundance on primordial Earth was water. However, the vast amounts of energy necessary to pull electrons from water presented a formidable obstacle to its utilization. Only when high‐energy solar radiation came to be employed in the process known as photosynthesis were "cyanobacteria" successful in linking the fixation of CO2 to hydrolysis [2]. Quinones were key to the evolution of photosystem I, yet another

While the increased access to electrons represented a potential windfall to energy‐starved cells, the development of photosynthesis was nonetheless catastrophic to life on Earth. Concomitant with the liberation of electrons from water by photosynthesis was the production of a new and

example of their power and adaptability in biological systems.

NAD+ or FAD+, reducing them to NADH and FADH2.

energy.

258 Vitamin K2 - Vital for Health and Wellbeing

biological systems.

The canonical pathway for menaquinone biosynthesis in bacteria is well‐established and has been reviewed in great detail elsewhere [7]. Of particular interest to this review, however, is the prenylation reaction mediated by MenA in which the lipophilic tail is attached to 1,4‐ dihydroxy‐2‐naphthoic acid (**Figure 2**). In essence, this is the critical step that links the redox‐ active quinone to the membrane. The lipophilic substrate of MenA is made up of repeating isoprenal subunits, the exact number of which is determined by the octaprenyl pyrophosphate (OPP) synthase encoded by the particular microbe. The ultimate chain length of these products is determined by a molecular ruler mechanism wherein bulky amino acid residues at the bottom of each of OPP's active sites block chain elongation [8], and it is this step that controls the identity of the primary MK produced by an organism. There is some evidence to suggest, however, that growth temperature also plays a role in the length and degree of saturation of the aliphatic side chain [9]. Phylloquinone biosynthesis in cyanobacteria is predicted to proceed via a pathway very similar to that of MK biosynthesis. However, the cyanobacterial MenA incorporates a mostly saturated phytyl tail at position C‐3 rather than the partially unsaturated isoprenyl side chain associated with MK [10]. Recently, an alternative pathway for menaquinone biosynthesis has been described in several Achaea and Gram‐negative bacteria, including *Helicobacter pylori*, Chlamydia species, and spirochetes [11]. While both pathways start with chorismate, the formation of the quinone proceeds via completely different reactions. The diversity of pathways for biosynthesis of MKs serves to underscore the

From Protein Folding to Blood Coagulation: Menaquinone as a Metabolic Link between Bacteria and Mammals

http://dx.doi.org/10.5772/63342

261

Phylloquinone biosynthesis in plants is not as well understood as in cyanobacteria, though the pathway likely mirrors that of menaquinone. One striking difference is that the first four reactions proceeding from chorismate, mediated by the products of the *menF*, *D*, *C*, and *H* genes in bacteria, are accomplished in plants by one fusion protein known as PHYLLO [12]. Later steps are compartmentalized between the chloroplast and peroxisome, adding an

Unlike the organisms mentioned above, higher‐order organisms are incapable of *de novo* synthesis of MKs. It is therefore imperative that MKs be supplied through diet, thus classifying MKs as a "vitamin" for mammals. The identification of MKs as essential vitamins arose from the Nobel Prize‐winning work of Edward Adelbert Doisy and Henrik Dam in 1943. Their recognition that a fat‐soluble compound played a key role in blood coagulation led them to name it "vitamin K" ("K" for the German word "Koagulationsvitamin"). Purification and characterization revealed two forms of vitamin K—phylloquinone thus became known as vitamin K1 and menaquinone as vitamin K2 with regards to their requirement in mammals. Though humans cannot synthesize vitamin K *de novo*, several homologs of menaquinone biosynthetic enzymes can be found in the human genome, including one for the prenylating enzyme MenA (UBIAD1). This enzyme was expressed in an insect cell line and was shown to be capable of converting menadione (K3) and K1 into MK‐4 [14]. Such a result suggested that while humans cannot make their own MKs, they may be capable of converting biosynthetic intermediates into the final product. Indeed, recent results show that the phytyl tail of K1 is cleaved in the intestine of mice to make K3 and then prenylated by UBIAD1 in the cerebrum [15, 16]. Furthermore, human subjects fed K1 exhibited increased levels of K3, indicating that dietary K1 may play a major role in overall MK levels [16], and gnotobiotic rats fed K1 saw increased levels of MK4 in tissues, indicating that the conversion is not dependent on gut bacteria [17]. UBIAD1 has also been demonstrated to be essential for the embryonic develop‐ ment of mice [18], so vitamin K's route through the body is not completely understood and

While the reactions requiring vitamin K in human metabolism are becoming clearer, the source of the vitamin K is still not completely understood. The presence of large numbers of bacteria in the human colon capable of synthesizing K2 would perhaps suggest that absorption of this bacterial byproduct might fulfill the human requirement. In fact, MK‐6 is made by *Eubacterium lentum*, MK‐7 by *Veillonella*, MK‐8 by *Enterobacteria*, and MK‐10 and MK‐11 are made by *Bacteroides* [19, 20]. The bacterial contribution to vitamin K pools in humans is supported by

importance of its role in metabolism.

additional level of complexity to production in plants [13].

the complexities begin with the vitamin's source.

**3. Sources of vitamin K**

**Figure 2.** Biosynthesis of menaquinone and phylloquinone. The highly conserved sequence of reactions required for conversion of chorismate to menaquinone is shown. The pathway for phylloquinone biosynthesis is thought to pro‐ ceed via the same steps, with the exception of the prenylation step mediated by MenA. The production of demethyl‐ phylloquinone versus demethylmenaquinone is determined by which substrate is provided to MenA by octaprenyl pyrophosphate synthase (OPP). A phytyl chain results in the production of demethylphylloquinone, whereas a more highly unsaturated isoprenyl chain results in demethylmenaquinone. In mice, metabolism of phylloquinone in the liv‐ er has been shown to release menadione, which in turn can be prenylated by the MenA homolog UBIAD1 to produce menaquinone.

for menaquinone biosynthesis has been described in several Achaea and Gram‐negative bacteria, including *Helicobacter pylori*, Chlamydia species, and spirochetes [11]. While both pathways start with chorismate, the formation of the quinone proceeds via completely different reactions. The diversity of pathways for biosynthesis of MKs serves to underscore the importance of its role in metabolism.

Phylloquinone biosynthesis in plants is not as well understood as in cyanobacteria, though the pathway likely mirrors that of menaquinone. One striking difference is that the first four reactions proceeding from chorismate, mediated by the products of the *menF*, *D*, *C*, and *H* genes in bacteria, are accomplished in plants by one fusion protein known as PHYLLO [12]. Later steps are compartmentalized between the chloroplast and peroxisome, adding an additional level of complexity to production in plants [13].

Unlike the organisms mentioned above, higher‐order organisms are incapable of *de novo* synthesis of MKs. It is therefore imperative that MKs be supplied through diet, thus classifying MKs as a "vitamin" for mammals. The identification of MKs as essential vitamins arose from the Nobel Prize‐winning work of Edward Adelbert Doisy and Henrik Dam in 1943. Their recognition that a fat‐soluble compound played a key role in blood coagulation led them to name it "vitamin K" ("K" for the German word "Koagulationsvitamin"). Purification and characterization revealed two forms of vitamin K—phylloquinone thus became known as vitamin K1 and menaquinone as vitamin K2 with regards to their requirement in mammals.

Though humans cannot synthesize vitamin K *de novo*, several homologs of menaquinone biosynthetic enzymes can be found in the human genome, including one for the prenylating enzyme MenA (UBIAD1). This enzyme was expressed in an insect cell line and was shown to be capable of converting menadione (K3) and K1 into MK‐4 [14]. Such a result suggested that while humans cannot make their own MKs, they may be capable of converting biosynthetic intermediates into the final product. Indeed, recent results show that the phytyl tail of K1 is cleaved in the intestine of mice to make K3 and then prenylated by UBIAD1 in the cerebrum [15, 16]. Furthermore, human subjects fed K1 exhibited increased levels of K3, indicating that dietary K1 may play a major role in overall MK levels [16], and gnotobiotic rats fed K1 saw increased levels of MK4 in tissues, indicating that the conversion is not dependent on gut bacteria [17]. UBIAD1 has also been demonstrated to be essential for the embryonic develop‐ ment of mice [18], so vitamin K's route through the body is not completely understood and the complexities begin with the vitamin's source.
