**4. The role of vitamin K2 in electron transport system**

Function of ubiquinone (Q) (coenzyme Q10) as a component of the mitochondrial respiratory chain in human is well established ("the chemiosmotic theory," Mitchell, 1978). In prokaryotes, especially in Gram‐positive bacteria, vitamin K2 (menaquinone) will transfer two electrons in a process of aerobic or anaerobic respiration. Respiration occurs in the cell membrane of prokaryotic cells. Electron donors transfer two electrons to menaquinone. Menaquinone in turn transfer these electrons to an electron acceptor. Schematic electron flow mediated by menaquinone in *M. tuberculosis* is illustrated in **Figure 2A**. The exact organization of enzymes in respiratory chains will vary among different bacteria. Nicotinamide adenine dinucleotide phosphate (NADH) is the most important electron donor in eukaryotes (**Figure 2B**); however, bacteria can use a number of different electron donors, dehydrogenases, oxidases and reductases, and electron acceptors. Electrons are transported along the membrane through menaquinone and a series of protein carriers.

is coupled with protons move through these complexes (**Figure 2**). Therefore, CoQ10 and menaquinone occupy a central and essential role in Adenosine triphosphate (ATP) synthesis [13, 14]. From the taxonomic studies, it is evident that the majority of Gram‐positive bacteria including *Mycobacterium* spp. utilize only menaquinone in their electron transport systems [15], and menaquinone biosynthesis is essential for survival of Gram‐positive bacteria [16, 17]. Several *in vitro* studies indicated that exogenous menaquinone did not rescue the bacteria treated with selective menaquinone biosynthesis inhibitors [18]. Menaquinone biosynthesis has been extensively studied in *E. coli*. A plethora of Gram‐negative organisms use CoQ in their electron transport systems when aerobic conditions prevail, but menaquinone under anaerobic conditions. However, the reaction chain facilitating electron transport humans does not use menaquinone. Unquestionably, the chain of reactions funneling the transport of electrons serves as a central component in the synthesis of Adenosine triphosphate (ATP) and the subsequent multiplication of bacteria (**Figure 2**). Hence, inhibitors of the biosynthesis of menaquinone or inhibitors specifically targeting the enzymes linked to electron transport systems display the potential for the development of novel and selective drugs against multidrug‐resistant (MDR) Gram‐positive bacteria. Although the functions of vitamin K1 in humans and vitamin K2 in bacteria are entirely different, drug discovery targeting vitamin K2 or its biosynthesis requires careful consideration of vitamin K distribution in tissue and selectivity against the target protein because essential vitamin K‐dependent protein(s) may be interfered by vitamin K biosynthesis inhibitors. Nonetheless, many evidences support that menaquinone biosynthesis inhibitors can be developed into selective antibacterial agents for

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infections caused by Gram‐positive bacteria and *Mycobacterium* spp.

Menaquinones play an important role in electron transport, and oxidative phosphorylation. In addition, they are responsible for active transport, and endospore formation in some *Bacillus* species [19]. The biosynthetic steps leading to menaquinone have been studied in *E. coli* (*vide supra*) [19, 20]. The synthesis of menaquinone is accomplished by MenA‐MenG as illustrated in **Figure 3**. These enzymes are encoded by two clusters of genes. The men gene cluster consists of the *MenB, C, D, E, and F* and a separate cluster containing *MenA* and *MenG* [21, 22]. The biosynthesis of menaquinone is initiated from chorismate and proceeds through a series of menaquinone‐specific reactions. MenF is isomerizing to chorismate in order to form isochorismate. MenD (a thiamine diphosphate‐dependent enzyme) catalyzes a Stetter‐like conjugate addition (a 1,4‐addition of an carbonyl molecule to α β‐unsaturated compound) of α‐ketoglutarate with isochorismate, forming 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohex‐ adiene‐1‐carboxylate. Its pyruvate moiety is eliminated by MenH to yield 2‐succinyl‐6‐ hydroxy‐2,4‐cyclohexadiene‐1‐carboxylate. MenC catalyzes aromatization of 2‐succinyl‐6‐ hydroxy‐2,4‐cyclohexadiene‐1‐carboxylate, forming *o*‐succinylbenzoate. MenE is an *o*‐ succinylbenzoate‐CoA ligase, which converts *o*‐succinylbenzoate to *o*‐succinylbenzoate‐CoA. Thereafter, MenB catalyzes a formal Dieckmann type of condensation of *o*‐succinylbenzoate‐ CoA to yield 1,4‐dihydroxy‐2‐naphthoyl‐CoA, which, in turn, is being hydrolyzed to 1,4‐

**5. Biosynthesis of menaquinone**

**Figure 2.** Schematic electron flow systems. (A) Electron transport systems of Gram‐positive bacterial and *Mycobacteri‐ um* spp. (B) Mitochondrial electron transport chain.

Protons are translocated across the cell membrane (from the cytoplasm to the periplasmic space) concomitantly. Synthesis of Adenosine triphosphate (ATP) from ADP and phosphate is coupled with protons move through these complexes (**Figure 2**). Therefore, CoQ10 and menaquinone occupy a central and essential role in Adenosine triphosphate (ATP) synthesis [13, 14]. From the taxonomic studies, it is evident that the majority of Gram‐positive bacteria including *Mycobacterium* spp. utilize only menaquinone in their electron transport systems [15], and menaquinone biosynthesis is essential for survival of Gram‐positive bacteria [16, 17]. Several *in vitro* studies indicated that exogenous menaquinone did not rescue the bacteria treated with selective menaquinone biosynthesis inhibitors [18]. Menaquinone biosynthesis has been extensively studied in *E. coli*. A plethora of Gram‐negative organisms use CoQ in their electron transport systems when aerobic conditions prevail, but menaquinone under anaerobic conditions. However, the reaction chain facilitating electron transport humans does not use menaquinone. Unquestionably, the chain of reactions funneling the transport of electrons serves as a central component in the synthesis of Adenosine triphosphate (ATP) and the subsequent multiplication of bacteria (**Figure 2**). Hence, inhibitors of the biosynthesis of menaquinone or inhibitors specifically targeting the enzymes linked to electron transport systems display the potential for the development of novel and selective drugs against multidrug‐resistant (MDR) Gram‐positive bacteria. Although the functions of vitamin K1 in humans and vitamin K2 in bacteria are entirely different, drug discovery targeting vitamin K2 or its biosynthesis requires careful consideration of vitamin K distribution in tissue and selectivity against the target protein because essential vitamin K‐dependent protein(s) may be interfered by vitamin K biosynthesis inhibitors. Nonetheless, many evidences support that menaquinone biosynthesis inhibitors can be developed into selective antibacterial agents for infections caused by Gram‐positive bacteria and *Mycobacterium* spp.

#### **5. Biosynthesis of menaquinone**

in respiratory chains will vary among different bacteria. Nicotinamide adenine dinucleotide phosphate (NADH) is the most important electron donor in eukaryotes (**Figure 2B**); however, bacteria can use a number of different electron donors, dehydrogenases, oxidases and reductases, and electron acceptors. Electrons are transported along the membrane through

**Figure 2.** Schematic electron flow systems. (A) Electron transport systems of Gram‐positive bacterial and *Mycobacteri‐*

Protons are translocated across the cell membrane (from the cytoplasm to the periplasmic space) concomitantly. Synthesis of Adenosine triphosphate (ATP) from ADP and phosphate

menaquinone and a series of protein carriers.

284 Vitamin K2 - Vital for Health and Wellbeing

*um* spp. (B) Mitochondrial electron transport chain.

Menaquinones play an important role in electron transport, and oxidative phosphorylation. In addition, they are responsible for active transport, and endospore formation in some *Bacillus* species [19]. The biosynthetic steps leading to menaquinone have been studied in *E. coli* (*vide supra*) [19, 20]. The synthesis of menaquinone is accomplished by MenA‐MenG as illustrated in **Figure 3**. These enzymes are encoded by two clusters of genes. The men gene cluster consists of the *MenB, C, D, E, and F* and a separate cluster containing *MenA* and *MenG* [21, 22]. The biosynthesis of menaquinone is initiated from chorismate and proceeds through a series of menaquinone‐specific reactions. MenF is isomerizing to chorismate in order to form isochorismate. MenD (a thiamine diphosphate‐dependent enzyme) catalyzes a Stetter‐like conjugate addition (a 1,4‐addition of an carbonyl molecule to α β‐unsaturated compound) of α‐ketoglutarate with isochorismate, forming 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohex‐ adiene‐1‐carboxylate. Its pyruvate moiety is eliminated by MenH to yield 2‐succinyl‐6‐ hydroxy‐2,4‐cyclohexadiene‐1‐carboxylate. MenC catalyzes aromatization of 2‐succinyl‐6‐ hydroxy‐2,4‐cyclohexadiene‐1‐carboxylate, forming *o*‐succinylbenzoate. MenE is an *o*‐ succinylbenzoate‐CoA ligase, which converts *o*‐succinylbenzoate to *o*‐succinylbenzoate‐CoA. Thereafter, MenB catalyzes a formal Dieckmann type of condensation of *o*‐succinylbenzoate‐ CoA to yield 1,4‐dihydroxy‐2‐naphthoyl‐CoA, which, in turn, is being hydrolyzed to 1,4‐ dihydroxy‐2‐naphthoate (DHNA) thioeter‐splitting enzyme encoded by *yfbB*. In contrast, the prenyl diphosphate with appropriate size (i.e., n = 7 in *E. coli*) is being biosynthesized by the iterative reaction of allyl diphosphate in the presence of isopentenyl diphosphate. Then, DHNA is prenylated and methylated by MenA and MenG, respectively, yielding menaqui‐ nones as the end product. The side chains of menaquinones vary in different species and even within the same organisms. The more common of menaquinones display 7, 8, and 9 isoprene (C‐5) units; MK‐7 serves as the major menaquinone entity in several Gram‐positive spore‐ forming bacteria. MK‐8 can be found in *E. coli*, while MK‐9 in common in *M. tuberculosis*. However, menaquinones containing 4, 5, 6, 10, 11, 12, and 13 isoprene units have been reported in bacteria.

Additionally, the MK/DMK ratio is not dependent on the fur locus, and substantial amounts of naphthoquinones (MK and DMK) are retrieved only during anaerobic conditions in *E. coli*. The menaquinones were detected almost no exception within the bacterial membrane. The total amount of naphthoquinones was found to be some 0.60 ∼ 1.09 μmol/g cell. It has become evident that several bacterial species do not have methylase (or MenG) and thus produce DMK as their sole quinone [19]. Conversion from MK to DMK is the last step in the biosynthesis. The activity of the DMK methylase is likely to be regulated by the presence or absence of the electron carriers or by the supply of *S*‐adenosylmethionine [20]. A bioinformatic analysis of whole‐genome sequences suggested that some microorganisms, including *Helicobacter pylori* and *Campylobacter jejuni*, and lactobacilli do not have orthologs of the men genes, although they synthesize menaquinone. These bacteria synthesize menaquinones in an alternative

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**6. Antibacterial drug discovery by targeting menaquinone biosynthesis**

Menaquinone is the sole quinone in the electron transport chain in the majority of Gram‐ positive bacteria including *Mycobacterium* spp. The biosynthetic pathway leading to menaqui‐ none is absent in humans; therefore, the bacterial enzymes responsible for menaquinone biosynthesis are potential drug targets for development of novel antibacterial agents. It is speculated that dormant (non‐replicating) *M. tuberculosis* displays a less active metabolism and also diminished energy reserves; however, Adenosine triphosphate (ATP) synthesis during oxidative phosphorylation is active during the dormant state. Therefore, inhibition of the menaquinone biosynthesis might exert serious effects on the maintenance of dormancy in *M. tuberculosis*. This concept emphasized the reports that phenothiazines block the type II Nicotinamide adenine dinucleotide phosphate (NADH): menaquinone oxidoreductase (**Figure 4**) in the bacterial respiratory chain and also were effective in killing non‐replicating *M. tuberculosis*[27]. Interestingly, it was demonstrated that inhibition of MenA (1,4‐dihydroxy‐ 2‐naphthoate prenyltransferase) (**Figure 3**) showed significant growth inhibitory activities against drug‐resistant *Mycobacterium* spp. and Gram‐positive bacteria [28]. MenA inhibitors

**Figure 4.** Electron flow in *M. tuberculosis* and type II Nicotinamide adenine dinucleotide phosphate (NADH) dehydro‐

genase inhibitors.

pathway via futalosine via alternative pathway (**Figure 3**) [23].

**Figure 3.** Biosynthesis of menaquinone.

Menaquinones are the predominant isoprenoid lipoquinones of Gram‐positive bacteria, whereas Gram‐negative bacteria and enterobacteria use menaquinone (MK), demethylmena‐ quinone (DMK), and ubiquinone (Q) in their electron transport chains (**Figure 2**). Recent studies have shown that several γ‐proteobacteria appear to share the similar electron transport system to that observed in *E. coli* [23–26]. Several studies indicated that the regulation of menaquinone biosynthesis of aerobically growing bacteria is different from those of bacteria under anaerobic respiratory conditions being controlled by FNR as the general regulator. Additionally, the MK/DMK ratio is not dependent on the fur locus, and substantial amounts of naphthoquinones (MK and DMK) are retrieved only during anaerobic conditions in *E. coli*. The menaquinones were detected almost no exception within the bacterial membrane. The total amount of naphthoquinones was found to be some 0.60 ∼ 1.09 μmol/g cell. It has become evident that several bacterial species do not have methylase (or MenG) and thus produce DMK as their sole quinone [19]. Conversion from MK to DMK is the last step in the biosynthesis. The activity of the DMK methylase is likely to be regulated by the presence or absence of the electron carriers or by the supply of *S*‐adenosylmethionine [20]. A bioinformatic analysis of whole‐genome sequences suggested that some microorganisms, including *Helicobacter pylori* and *Campylobacter jejuni*, and lactobacilli do not have orthologs of the men genes, although they synthesize menaquinone. These bacteria synthesize menaquinones in an alternative pathway via futalosine via alternative pathway (**Figure 3**) [23].

dihydroxy‐2‐naphthoate (DHNA) thioeter‐splitting enzyme encoded by *yfbB*. In contrast, the prenyl diphosphate with appropriate size (i.e., n = 7 in *E. coli*) is being biosynthesized by the iterative reaction of allyl diphosphate in the presence of isopentenyl diphosphate. Then, DHNA is prenylated and methylated by MenA and MenG, respectively, yielding menaqui‐ nones as the end product. The side chains of menaquinones vary in different species and even within the same organisms. The more common of menaquinones display 7, 8, and 9 isoprene (C‐5) units; MK‐7 serves as the major menaquinone entity in several Gram‐positive spore‐ forming bacteria. MK‐8 can be found in *E. coli*, while MK‐9 in common in *M. tuberculosis*. However, menaquinones containing 4, 5, 6, 10, 11, 12, and 13 isoprene units have been reported

Menaquinones are the predominant isoprenoid lipoquinones of Gram‐positive bacteria, whereas Gram‐negative bacteria and enterobacteria use menaquinone (MK), demethylmena‐ quinone (DMK), and ubiquinone (Q) in their electron transport chains (**Figure 2**). Recent studies have shown that several γ‐proteobacteria appear to share the similar electron transport system to that observed in *E. coli* [23–26]. Several studies indicated that the regulation of menaquinone biosynthesis of aerobically growing bacteria is different from those of bacteria under anaerobic respiratory conditions being controlled by FNR as the general regulator.

in bacteria.

286 Vitamin K2 - Vital for Health and Wellbeing

**Figure 3.** Biosynthesis of menaquinone.
