**3. Sources of vitamin K**

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

260 Vitamin K2 - Vital for Health and Wellbeing

**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.

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 studies done with gnotobiotic rats fed vitamin K‐free diets. The rapidly developing hemor‐ rhagic conditions in these rats could be reversed by supplying bacteria from conventionally raised rats, suggesting that absorption from the bowel provided sufficient quantities of K2 [21]. Concordant with this is the observation that taking broad‐spectrum antibiotics can reduce vitamin K production by more than 70% [22]. However, K2 is embedded within the bacterial inner membrane, and as such would appear to be inaccessible to passive absorption. MKs have been shown to be secreted by some organisms [23], and it is also possible that water‐soluble precursors of MK biosynthesis might be more readily available [7]. However, this scenario is further complicated by the fact that there is very little evidence that the large intestine is capable of absorption of MKs. Uptake has been shown to be poor in rats [24] and infants [25]. Even the finding that antibiotic treatment lowered vitamin K production does not conclusively identify bacteria as a major source of human vitamin K2 pools, as some antibiotics have been shown to inhibit the human enzymes necessary for recycling vitamin K2 [26]. The role of the micro‐ biome in the production of K2 is therefore questionable and would suggest that perhaps vitamin K stores in humans might be the result of dietary intake.

should therefore be very similar, a fact underlined by the nearly identical mid‐point redox potentials as determined by voltammetry [31, 32] (**Figure 3**). The degree of lipophilicity in the tails most likely dictates mobility of the quinones in the membrane, with the partially saturated isoprenyl tail of MK allowing for greater freedom of movement compared to the mostly unsaturated chain of K1. Additionally, longer chain MKs are likely stiffer and more viscous in the membrane due to the greater surface areas available for van der Waals interactions. For these reasons, the preferential incorporation of one MK over another into a redox‐active enzyme is most likely due to availability within the membrane as well as the ability of the enzyme to accommodate different length side chains. In microsomal fractions, MK2 and MK3 were shown to have much higher activities than K1 [33], while a partially purified enzymatic system showed similar activities for MK2‐6 compared to K1. MKs with seven or more isopre‐

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

Vitamin K2 has been found to play a role in protection against oxidative stress and inflamma‐ tion in mammals [35], and improved locomotion defects in mutant fruit flies [36], suggesting that it might benefit human patients suffering mitochondrial pathologies. Mounting evidence suggests that MK‐4 is an important component of sphingolipid biosynthesis and can inhibit the proliferation of several cancer cell lines [37]. The exact role of vitamin K2 in these processes is unknown however—its most thoroughly understood use is in protein modification.

Numerous proteins in vertebrates are modified post‐translationally as a means of regulating and enhancing their activity. One such modification is the carboxylation of glutamate residues within Gla domains, which is mediated by the enzyme gamma‐glutamyl carboxylase (GGCX). This modification allows for the high‐affinity binding of calcium ions, which in turn mediates a conformational change necessary for proper folding of the protein. Gla‐containing proteins play important roles in the venom of snakes and the toxins of cone snails [38], and they have numerous functions in humans including bone development, calcification, and sphingolipid metabolism [35, 39]. The cell‐signaling activities of the vitamin K‐dependent proteins Gas6 and protein S may also be crucial to cognitive processes [35]. Among the Gla‐containing proteins, however, those involved in blood coagulation have received the most attention. Carboxylation of several of these factors activates them and thereby sets off a cascade leading to clotting. The

) can be accepted or donated in step‐wise

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noid units were not as active [34].

transfers from partner proteins.

**Figure 3.** Resonance structures of napthoquinone species. Two electrons (e‐

Low concentrations of vitamin K2 can be found in dairy, meat, and fermented foods like natto [27], but makes up only 10% of total dietary vitamin K intake. While K1, found in a variety of green leafy plants and vegetable oils, is present in much higher amounts, it is not readily absorbed in the intestines as it is strongly bound to vegetable fiber [28]. Vitamin K is not transported by specific plasma carrier proteins like other fat‐soluble vitamins, but is instead shuttled by lipoproteins. The small fraction of K1 that is absorbed is almost exclusively incorporated into the triacylglycerol‐rich lipoprotein (TGRLP) fraction, while dietary K2 is associated with low‐density lipoprotein (LDL) fraction [29]. These divergent pathways would deliver large amounts of K1 to the liver, but efficient delivery to extrahepatic tissues would only occur for K2. Measurements of the concentrations of vitamins K in various tissues mostly back this up, showing that K1 levels are low in the brain, kidneys, and lungs but high in the liver, heart, and pancreas; K2 (in the form of MK‐4) was found to be in high concentration in the brain, kidneys, and pancreas but in low concentration in the liver, heart, and lungs. As for longer chain K2s, MK6‐11 were found in the liver and trace amounts of MK6‐9 were found in the heart and pancreas [30]. MK10 and MK11 may be major contributors to the hepatic pool of K2 [26], and the presence of these long‐chain MKs again raise the possibility that the commensal population of colonic bacteria may somehow contribute to overall vitamin K levels in the host, as analysis of tissue samples has only shown the ability to synthesize MK‐4 from K1. However, the presence of potential homologs for other prenyl diphosphate synthases in the genome further suggests that humans may be capable of producing longer chain MKs as well. Overall the data clearly indicate that dietary K1 is a major contributor to vitamin K levels in the body, but a full accounting of its sources has yet to emerge.
