**3. Structure of lactoperoxidase**

Lactoperoxidase folds into an oval-shaped structure which is largely -helical with 20 helices and two small anti-parallel β-strands (Figure 1) (Singh et al., 2008). The central core of the protein consists of five long -helices, H2, H5, H6, H8 and H12. The N-terminal

Mammalian Heme Peroxidases and *Mycobacterium tuberculosis* 281

The heme moiety in LPO is a derivative of protoporphyrin IX (Thanabal & La Mar, 1989) in which the methyl groups on pyrrole rings A and C are modified to allow formation of ester

linkages with carboxylic groups of Glu258 and Asp108 respectively (Figure 2).

Fig. 2. The structure of the heme moiety showing a standard nomenclature. The two

The ferric iron atom is coordinated to four heme nitrogen atoms in a slightly distorted planar arrangement. The fifth coordination is provided by proximal His351 while on the sixth side a conserved water molecule W1 is located at a hydrogen bonded distance from the heme iron. The heme moiety is deeply buried inside the protein molecule while the heme cavity is surrounded by a number of -helices from three sides. The two β-strands, S1 and S2 are situated on the upper side of the opening to the heme cavity (Figure 1). Overall, the plane of heme protoporphyrin IX moiety is slightly distorted from planarity. The pyrrole rings A, C and D are essentially planar while pyrrole ring B is slightly distorted from planarity. The iron position is shifted slightly towards the proximal side. The carboxyl group of the pyrrole ring D propionate interacts with the guanidinium groups of Arg348 and Arg440. In contrast, the ring C propionate interacts with Asp112 Oδ2, Ala112 N and a

The substrate binding site is formed on the distal heme side. In the native structure of LPO, the substrate-binding space is occupied by six water molecules W1, W2, W3, W4¸ W5 and W6 (Figure 3). In the resting state, W1 is linked to ferric iron at a hydrogen bonded distance.

covalent linkages to protein are indicated.

water molecule.

**3.2 Substrate specificity** 

**3.1 Heme moiety** 

segment of LPO does not form any repetitive structure till amino acid residue number 75 from where -helix H1 starts which is a short helix. It is connected to H2 through a long chain. The -helix H2 is connected to two unique -helices H2a and H2b which are absent in MPO (Zeng & Fenna, 1992). These helices are followed by two short -helices, H3 and H4. The -helices H5 and H6 are connected through a V-shaped loop which is flanked by two short extended chains. A core -helix H8 forms a triangle with helices H5 and H6. Heme group is sandwiched between helices H2 and H8. This is connected to the region consisting of helices H12, H13, H14, H15, H16 and parts of helices H17, H18 and H19. This region represents the crown of the back face of the core region. Three helices H2, H5 and H6 also form a triangle below which lies the heme group. The other two core -helices, H8 and H12 which run parallel to each other and form the lower wall on which the heme moiety rests. The heme moiety is located nearly at the centre of the protein. The δ-heme side is accessible through a channel from the surface of the protein. The structure is stabilized by a calcium ion which forms a seven fold co-ordination. In the structure of LPO, Ser-198 is phosphorylated and seems to facilitate the entry of calcium ion into the core of protein molecule.

Fig. 1. Molecular structure of lactoperoxidase. Cylinders indicate -helices which are labelled. -strands are indicated as arrows. Heme group is shown in the centre as a ball and stick model. The positions of four glycan moieties are also indicated.

### **3.1 Heme moiety**

280 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

segment of LPO does not form any repetitive structure till amino acid residue number 75 from where -helix H1 starts which is a short helix. It is connected to H2 through a long chain. The -helix H2 is connected to two unique -helices H2a and H2b which are absent in MPO (Zeng & Fenna, 1992). These helices are followed by two short -helices, H3 and H4. The -helices H5 and H6 are connected through a V-shaped loop which is flanked by two short extended chains. A core -helix H8 forms a triangle with helices H5 and H6. Heme group is sandwiched between helices H2 and H8. This is connected to the region consisting of helices H12, H13, H14, H15, H16 and parts of helices H17, H18 and H19. This region represents the crown of the back face of the core region. Three helices H2, H5 and H6 also form a triangle below which lies the heme group. The other two core -helices, H8 and H12 which run parallel to each other and form the lower wall on which the heme moiety rests. The heme moiety is located nearly at the centre of the protein. The δ-heme side is accessible through a channel from the surface of the protein. The structure is stabilized by a calcium ion which forms a seven fold co-ordination. In the structure of LPO, Ser-198 is phosphorylated and seems to facilitate the entry of calcium ion into the core of protein

Fig. 1. Molecular structure of lactoperoxidase. Cylinders indicate -helices which are labelled. -strands are indicated as arrows. Heme group is shown in the centre as a ball and

stick model. The positions of four glycan moieties are also indicated.

molecule.

The heme moiety in LPO is a derivative of protoporphyrin IX (Thanabal & La Mar, 1989) in which the methyl groups on pyrrole rings A and C are modified to allow formation of ester linkages with carboxylic groups of Glu258 and Asp108 respectively (Figure 2).

Fig. 2. The structure of the heme moiety showing a standard nomenclature. The two covalent linkages to protein are indicated.

The ferric iron atom is coordinated to four heme nitrogen atoms in a slightly distorted planar arrangement. The fifth coordination is provided by proximal His351 while on the sixth side a conserved water molecule W1 is located at a hydrogen bonded distance from the heme iron. The heme moiety is deeply buried inside the protein molecule while the heme cavity is surrounded by a number of -helices from three sides. The two β-strands, S1 and S2 are situated on the upper side of the opening to the heme cavity (Figure 1). Overall, the plane of heme protoporphyrin IX moiety is slightly distorted from planarity. The pyrrole rings A, C and D are essentially planar while pyrrole ring B is slightly distorted from planarity. The iron position is shifted slightly towards the proximal side. The carboxyl group of the pyrrole ring D propionate interacts with the guanidinium groups of Arg348 and Arg440. In contrast, the ring C propionate interacts with Asp112 Oδ2, Ala112 N and a water molecule.

### **3.2 Substrate specificity**

The substrate binding site is formed on the distal heme side. In the native structure of LPO, the substrate-binding space is occupied by six water molecules W1, W2, W3, W4¸ W5 and W6 (Figure 3). In the resting state, W1 is linked to ferric iron at a hydrogen bonded distance.

Mammalian Heme Peroxidases and *Mycobacterium tuberculosis* 283

Fig. 4. Hydrogen bonded chain involving His351, Fe3+, W1, His109, W2, His266, W3, W4, W5 and W6 where W1 is hydrogen bonded to Fe3+ and W6 is located near the protein surface.

Fig. 5. A schematic representation of the diffusion channel and substrate-binding site in LPO

The substrate diffusion channel in LPO is long and surrounded by aromatic residues from all sides. As a result it allows the diffusion of small aromatic compounds from surface to the substrate binding site on the distal heme side. The aromatic compounds such as acetyl

drawn using PDB-ID: 3GC1.

**3.3 Specificity for aromatic compounds** 

When H2O2 is supplied, it expels W1 and forms the sixth coordination. On this side, His109 works as proton donor-acceptor residue as it is linked to a chain of water molecules that facilitate proton relay (Figure 4). When ligands bind to LPO in the substrate binding site on the distal heme side, it plays an essential role in the enzymatic action. The substrate-binding site is surrounded by heme moiety on one side while residues, His109, Phe113, Phe254 and Arg255 occupy the opposite side. The front end of the site consists of Gln105 while Glu258 supports it from below. The other wall is made up of residues, Phe381, Phe422, Gln423 and Pro424. The substrate-binding site is connected by a long channel formed by hydrophobic aromatic residues including Pro234, Pro236, Phe380, Phe381 and Phe254 on one side while Leu421, Phe422, Gln423 and Pro424 on the opposite (Figure 5). The length of substrate diffusion channel in LPO is approximately 22Å while its diameter is about 10Å. The substrate-binding site is connected to the surface of the protein through this diffusion channel.

Fig. 3. The structure of the substrate-binding site on the distal heme side in LPO and the positions of six conserved water molecules, W1, W2, W3, W4¸ W5 and W6 as observed in the unliganded structure. The proximal and distal sites have been indicated.

When H2O2 is supplied, it expels W1 and forms the sixth coordination. On this side, His109 works as proton donor-acceptor residue as it is linked to a chain of water molecules that facilitate proton relay (Figure 4). When ligands bind to LPO in the substrate binding site on the distal heme side, it plays an essential role in the enzymatic action. The substrate-binding site is surrounded by heme moiety on one side while residues, His109, Phe113, Phe254 and Arg255 occupy the opposite side. The front end of the site consists of Gln105 while Glu258 supports it from below. The other wall is made up of residues, Phe381, Phe422, Gln423 and Pro424. The substrate-binding site is connected by a long channel formed by hydrophobic aromatic residues including Pro234, Pro236, Phe380, Phe381 and Phe254 on one side while Leu421, Phe422, Gln423 and Pro424 on the opposite (Figure 5). The length of substrate diffusion channel in LPO is approximately 22Å while its diameter is about 10Å. The substrate-binding site is connected to the surface of the protein through this diffusion

**Fe3+**

**His 351**

**Heme**

Proximal side

**His 109**

**Phe 113**

**Gln 105**

**W 1**

**W**2

**Arg 255**

**W**3

Distal side

**Phe**-**254**

the unliganded structure. The proximal and distal sites have been indicated.

Fig. 3. The structure of the substrate-binding site on the distal heme side in LPO and the positions of six conserved water molecules, W1, W2, W3, W4¸ W5 and W6 as observed in

**W 5**

**W**4

**W**6

**Glu 258**

**Gln 423**

**Phe 381**

**Pro 424**

**Phe 422**

channel.

Fig. 4. Hydrogen bonded chain involving His351, Fe3+, W1, His109, W2, His266, W3, W4, W5 and W6 where W1 is hydrogen bonded to Fe3+ and W6 is located near the protein surface.

Fig. 5. A schematic representation of the diffusion channel and substrate-binding site in LPO drawn using PDB-ID: 3GC1.

#### **3.3 Specificity for aromatic compounds**

The substrate diffusion channel in LPO is long and surrounded by aromatic residues from all sides. As a result it allows the diffusion of small aromatic compounds from surface to the substrate binding site on the distal heme side. The aromatic compounds such as acetyl

Mammalian Heme Peroxidases and *Mycobacterium tuberculosis* 285

equations 1 to 3. This product is similar to that produced by *Mycobacterium tuberculosis*

**Phe 254**

Fig. 7. Showing binding of INH to LPO as a substrate. The dotted lines indicate hydrogen

As revealed by binding studies using surface Plasmon resonance technique, pyrazinamide (PZA) has also been found to bind to LPO with a slightly lower affinity than that of INH (dissociation constant, Kd = 1.2 10-5 M). The structure determination of PZA-bound LPO revealed that it occupies a position in the centre of the substrate-binding site on the distal heme side. Upon binding to pyrazinamide, three water molecules, W4, W5 and W6 were expelled from the substrate-binding site. It retained three water molecules, W1, W2 and W3. The nearest nitrogen atom of PZA is about 5.5Å away from the oxygen atom of conserved water molecule W1. PZA and conserved water molecule W1 are separated from each other by another water molecule W2. The carboxamide nitrogen atom of PZA forms a hydrogen bond with W2 which in turn is hydrogen bonded at W1 (Figure 8). It reflects a slightly weaker affinity of PZA towards the position of W1. However, when H2O2 is introduced, it is expected to move closer to H2O2 and the product may be formed. The position occupied by PZA in the substrate-binding site appears to be suitable for the

catalytic action by LPO. However, the nature of product is not characterized clearly.

*Mycobacterium tuberculosis* catalase peroxidase (*Mt*CP) is a dimeric bi-functional hemedependent enzyme of molecular mass of 160 kDa. Its primary function is of catalase activity. However, its role as a peroxidase is well established and its peroxidative activity is comparable with those of other mono-functional heme peroxidases (Metcalfe et al., 2008;

**Phe 381**

**W**3′

**Isoniazid**

**Fe3+**

**Arg 255**

**W 1**

**Heme**

**His 351**

**His 109**

catalase peroxidase (*Mt*CP) (Bertrand et al., 2004).

**Gln 423**

**Pro 424**

**3.5 Complex of LPO with Pyrazinamide** 

**3.6 Structure of catalase peroxidase** 

bonds.

**Phe 422**

salicylic acid (ASA) and salicylhydroxamic acid (SHA) have been shown to bind to LPO at the substrate-binding site (Figure 6) (Singh et al., 2009). It has been observed that the aromatic compounds which bind to LPO but do not expel the conserved water molecule, W1 from its original position as has been observed in the case of acetyl salicylic acid act as substrates (Figure 6A) while those that expel the conserved water molecule, W1 and coordinate directly with the heme iron act as inhibitors as found in the case of salicylhydroxamic acid (Figure 6B). Therefore, the mode of binding of substrates and inhibitors differ in terms of their positioning in the substrate-binding site.

Fig. 6. (A) Acetyl salicylic acid (ASA) bound to LPO as a substrate at the substrate-binding site (PDB-ID: 3GCL). The hydrogen bonded interactions are indicated by dotted lines. (B) Salicylhydroxamic acid (SHA) bound to LPO as an inhibitor in the substrate-binding site (PDB-ID: 3GCJ). The hydrogen bonded interactions are indicated by dotted lines.

#### **3.4 Structure of the LPO complex with Isoniazid**

Since LPO has been shown to bind to small aromatic compounds, the binding properties of LPO with INH were examined using surface plasmon resonance technique. The protein molecule was immobilized on the chip while INH was used as a solution in a mobile phase. These measurements gave a value of 1.1 10-6 M for the dissociation constant (Kd) for the binding of LPO with INH. In order to determine the interactions between LPO and INH, the crystal structure of the INH-bound LPO was determined (Singh et al., 2010). The structure of the LPO-INH complex showed that INH upon binding to LPO in the substrate-binding site on the distal heme side expelled four water molecules W2, W4, W5 and W6 from the substrate-binding site on the distal heme side. Two out of six conserved water molecules, W1 and W3 remained unperturbed. INH binds to LPO with an appropriate orientation as its pyridine ring nitrogen atom is at a hydrogen bonded distance from the conserved water molecule W1 (Figure 7). Therefore, when H2O2 is introduced into the solution containing LPO and INH, it expels the water molecule W1 and the reaction gets initiated. The product isonicotinyl radical is formed as described by

salicylic acid (ASA) and salicylhydroxamic acid (SHA) have been shown to bind to LPO at the substrate-binding site (Figure 6) (Singh et al., 2009). It has been observed that the aromatic compounds which bind to LPO but do not expel the conserved water molecule, W1 from its original position as has been observed in the case of acetyl salicylic acid act as substrates (Figure 6A) while those that expel the conserved water molecule, W1 and coordinate directly with the heme iron act as inhibitors as found in the case of salicylhydroxamic acid (Figure 6B). Therefore, the mode of binding of substrates and

**(A) (B)** Fig. 6. (A) Acetyl salicylic acid (ASA) bound to LPO as a substrate at the substrate-binding site (PDB-ID: 3GCL). The hydrogen bonded interactions are indicated by dotted lines. (B) Salicylhydroxamic acid (SHA) bound to LPO as an inhibitor in the substrate-binding site (PDB-ID: 3GCJ). The hydrogen bonded interactions are indicated by dotted lines.

Since LPO has been shown to bind to small aromatic compounds, the binding properties of LPO with INH were examined using surface plasmon resonance technique. The protein molecule was immobilized on the chip while INH was used as a solution in a mobile phase. These measurements gave a value of 1.1 10-6 M for the dissociation constant (Kd) for the binding of LPO with INH. In order to determine the interactions between LPO and INH, the crystal structure of the INH-bound LPO was determined (Singh et al., 2010). The structure of the LPO-INH complex showed that INH upon binding to LPO in the substrate-binding site on the distal heme side expelled four water molecules W2, W4, W5 and W6 from the substrate-binding site on the distal heme side. Two out of six conserved water molecules, W1 and W3 remained unperturbed. INH binds to LPO with an appropriate orientation as its pyridine ring nitrogen atom is at a hydrogen bonded distance from the conserved water molecule W1 (Figure 7). Therefore, when H2O2 is introduced into the solution containing LPO and INH, it expels the water molecule W1 and the reaction gets initiated. The product isonicotinyl radical is formed as described by

**3.4 Structure of the LPO complex with Isoniazid** 

inhibitors differ in terms of their positioning in the substrate-binding site.

equations 1 to 3. This product is similar to that produced by *Mycobacterium tuberculosis* catalase peroxidase (*Mt*CP) (Bertrand et al., 2004).

Fig. 7. Showing binding of INH to LPO as a substrate. The dotted lines indicate hydrogen bonds.

#### **3.5 Complex of LPO with Pyrazinamide**

As revealed by binding studies using surface Plasmon resonance technique, pyrazinamide (PZA) has also been found to bind to LPO with a slightly lower affinity than that of INH (dissociation constant, Kd = 1.2 10-5 M). The structure determination of PZA-bound LPO revealed that it occupies a position in the centre of the substrate-binding site on the distal heme side. Upon binding to pyrazinamide, three water molecules, W4, W5 and W6 were expelled from the substrate-binding site. It retained three water molecules, W1, W2 and W3. The nearest nitrogen atom of PZA is about 5.5Å away from the oxygen atom of conserved water molecule W1. PZA and conserved water molecule W1 are separated from each other by another water molecule W2. The carboxamide nitrogen atom of PZA forms a hydrogen bond with W2 which in turn is hydrogen bonded at W1 (Figure 8). It reflects a slightly weaker affinity of PZA towards the position of W1. However, when H2O2 is introduced, it is expected to move closer to H2O2 and the product may be formed. The position occupied by PZA in the substrate-binding site appears to be suitable for the catalytic action by LPO. However, the nature of product is not characterized clearly.

#### **3.6 Structure of catalase peroxidase**

*Mycobacterium tuberculosis* catalase peroxidase (*Mt*CP) is a dimeric bi-functional hemedependent enzyme of molecular mass of 160 kDa. Its primary function is of catalase activity. However, its role as a peroxidase is well established and its peroxidative activity is comparable with those of other mono-functional heme peroxidases (Metcalfe et al., 2008;

Mammalian Heme Peroxidases and *Mycobacterium tuberculosis* 287

Fig. 9. Schematic representation of the diffusion channel and substrate-binding site in *Mt*CP (PDB-ID: 1SJ2). Ser315 as Gln423 in LPO is involved in the interaction with INH while

Fig. 10. Schematic representation for the binding site in PncA (PDB-ID: 3PL1) showing

significant similarities to those of LPO and *Mt*CP.

His108 as His109 in LPO is involved in the catalytic mechanism.

Fig. 8. Binding of PZA to LPO as a substrate in the substrate-binding site (PDB-ID: XYZ). The dotted lines indicate hydrogen bonded interactions.

Pierattelli et al., 2004). Sofar, crystal structure of only unliganded-*Mt*CP has been determined (Bertrand et al., 2004). However, modeling and computational studies have indicated the binding of INH at the δ-meso heme edge which superseded the existing proposition of it being in a surface loop structure of the enzyme (Mo et al., 2004). Yet another important observation pertains to the role of residue Ser315 in the binding of INH. It was suggested that Ser315 might interact with INH but it would not be involved in the catalytic action. It was further indicated that His108 on the distal heme side was involved in enzyme catalytic action (Figure 9). A comparison of the binding of INH with LPO where Gln423 forms a hydrogen bond with amino nitrogen atom of the hydrazide moiety. However, it is not responsible for the catalytic action as it is important for promoting an appropriate orientation of the substrate. On the other hand, it has been shown that His109 plays the role of proton donor/acceptor as it forms two hydrogen bonds, one with pyridine ring nitrogen atom of INH and another with conserved water molecule W1. These modes of binding of INH show a striking similarity in the substrate-binding sites on the distal heme side of the two enzymes, *Mt*CP and LPO indicating similar mechanisms of actions.

#### **3.7 Complex of pyrazinamidase with PZA**

Pyrazinamidase (PncA) activates PZA into POA. The crystal structure of *Mycobacterium tuberculosis* pyrazinamidase has been determined (Petrella et al., 2011) which shows that PncA folds into an /β single domain protein. It has an iron binding site involving residues Asp49, His51, His57 and His71 and consists of a catalytic triad with residues Cys138, Asp8 and Lys96. The substrate binding cavity in pyrazinamidase is a part of the cleft which is shown schematically in Figure 10. The amino acid residues of the active site are located on

**Fe3+**

**W1**

**His 109**

**His 351**

**Arg 255**

**W**2

**W**3

**Gln 423**

**Phe 254**

Fig. 8. Binding of PZA to LPO as a substrate in the substrate-binding site (PDB-ID: XYZ).

side of the two enzymes, *Mt*CP and LPO indicating similar mechanisms of actions.

Pyrazinamidase (PncA) activates PZA into POA. The crystal structure of *Mycobacterium tuberculosis* pyrazinamidase has been determined (Petrella et al., 2011) which shows that PncA folds into an /β single domain protein. It has an iron binding site involving residues Asp49, His51, His57 and His71 and consists of a catalytic triad with residues Cys138, Asp8 and Lys96. The substrate binding cavity in pyrazinamidase is a part of the cleft which is shown schematically in Figure 10. The amino acid residues of the active site are located on

Pierattelli et al., 2004). Sofar, crystal structure of only unliganded-*Mt*CP has been determined (Bertrand et al., 2004). However, modeling and computational studies have indicated the binding of INH at the δ-meso heme edge which superseded the existing proposition of it being in a surface loop structure of the enzyme (Mo et al., 2004). Yet another important observation pertains to the role of residue Ser315 in the binding of INH. It was suggested that Ser315 might interact with INH but it would not be involved in the catalytic action. It was further indicated that His108 on the distal heme side was involved in enzyme catalytic action (Figure 9). A comparison of the binding of INH with LPO where Gln423 forms a hydrogen bond with amino nitrogen atom of the hydrazide moiety. However, it is not responsible for the catalytic action as it is important for promoting an appropriate orientation of the substrate. On the other hand, it has been shown that His109 plays the role of proton donor/acceptor as it forms two hydrogen bonds, one with pyridine ring nitrogen atom of INH and another with conserved water molecule W1. These modes of binding of INH show a striking similarity in the substrate-binding sites on the distal heme

**Pyrazinamide**

**Pro 424**

**Phe 422**

**Phe 381**

**3.7 Complex of pyrazinamidase with PZA** 

The dotted lines indicate hydrogen bonded interactions.

Fig. 9. Schematic representation of the diffusion channel and substrate-binding site in *Mt*CP (PDB-ID: 1SJ2). Ser315 as Gln423 in LPO is involved in the interaction with INH while His108 as His109 in LPO is involved in the catalytic mechanism.

Fig. 10. Schematic representation for the binding site in PncA (PDB-ID: 3PL1) showing significant similarities to those of LPO and *Mt*CP.

Mammalian Heme Peroxidases and *Mycobacterium tuberculosis* 289

Medical Research, New Delhi and Council of Scientific and Industrial Research, New Delhi for the award of fellowships. TPS thanks Department of Biotechnology (DBT) for the award

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Dolphin, D., Muljiani, Z., Rousseau, K., Borg, D. C., Fajer, J., & Felton, R. H. (1973). The

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**6. References** 

5393

one of the cleft. Although crystal structure of PZA-bound PncA is not known but it is suggested that the binding of PZA involves hydrogen bonded interaction between pyridyl nitrogen atom of PZA and the conserved water molecule W1 which is coordinated to Fe2+ ion. Another possible interaction is provided by carbonyl oxygen atom of PZA with peptide Ala134 – Cys138. Overall, the role played by Fe2+ ion and His57 is critical in stabilizing the structure of substrate-binding site. Finally the conversion of PZA to POA and its accumulation results in lowering the intracellular pH to suboptimal level and thus causing the inactivation of some of the critically important proteins such as fatty acid synthase resulting in the killing of bacteria (Zimhony et al., 2000).
