**Mammalian Heme Peroxidases and**  *Mycobacterium tuberculosis*

Amit K. Singh, Nisha Pandey, Mau Sinha, Sujata Sharma and Tej P. Singh *Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India* 

#### **1. Introduction**

276 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

Young D, Lathigra R, Hendrix R, Sweetser D, Young RA. (1988). Stress proteins are immune

Tuberculosis (TB) is a lethal infectious disease which is caused by *Mycobacterium tuberculosis*. The alarming rate at which the incidence of bacterial resistance to known antibiotics has been rising is a serious cause of concern. At present, the two well known anti-tuberculosis drugs, isonicotinic acid hydrazide (INH, isoniazid) and pyrazinamide (PZA, pyrazin-2 carboxamide) which are important components of the current course of the first-line TB chemotherapy suffer from increasing bacterial resistance. The other drugs of the combination therapy include rifampicin and ethambutol. It may be noted that both INH and PZA are prodrugs and require specific enzymes to convert them into drugs. INH is activated by a bacterial heme enzyme catalase peroxidase (*Mt*CP) into a free radical form (Scheme I) (Zhang et al., 1992). The structure of unliganded *Mt*CP is known (Bertrand et al., 2004) and detailed information is available about the substrate-binding site and the residues that might be involved in the binding and conversion of INH into a beneficial product. However, a precise mode of binding and the mechanism of action are not yet clearly understood because the structure of INH bound *Mt*CP is not yet determined. On the other hand, PZA is metabolized into its active form pyrazinoic acid (POA) by amidase activity of the *Mycobacterium tuberculosis* nicotinamidase/pyrazinamidase (PncA) (Scheme II) (Konno et al., 1967). Although the crystal structure of pyrazinamidase in complex with POA is known but the structure of the complex with the original compound PZA is not yet determined. Therefore, the mode of binding of PZA with PncA has not so far been revealed. As shown by the crystal structure of the complex of LPO with INH, the binding of INH to lactoperoxidase (LPO) occurs through the distal heme cavity where INH interacts with a conserved water molecule W1 which is hydrogen bonded to ferric iron (Singh et al., 2010). Similarly, as revealed by the structure determination of the complex formed between LPO and PZA, PZA has been located in the substrate-binding site and interacts with substrate recognition residues of LPO (PDB ID: 3R4X) indicating a possible role of LPO in the conversion of PZA into an active form. Although the crystal structure of the PZA bound PncA is not known but a piece of information is available on the possible mode of ligand binding based on the molecular modeling data (Petrella et al., 2011). Therefore, it is of great interest that both prodrugs, INH and PZA bind to LPO specifically at the substrate-binding site on the distal heme side as the substrates bind to LPO (Singh et al., 2009) so that these

Mammalian Heme Peroxidases and *Mycobacterium tuberculosis* 279

primary function of LPO is to catalyze the bielectronic oxidation of pseudohalide (SCN- ion)

The biological significance of lactoperoxidase is related to its involvement in the natural host defense system against invading micro-organisms (Reiter & Harnulv, 1984; Reiter & Perraudin, 1991; Wolfson & Sumner, 1993). Apart from that, it was also reported to be involved in the antiviral activity (Mikola et al., 1995; Pourtois et al., 1990; Shin et al., 2005), degradation of various carcinogens and protection of animal cells against peroxidative effects (Tenovuo et al, 1985). It may be noted that the reaction products generated by the catalytic

Lactoperoxidase is a heme-containing single chain protein with 595 amino acid residues. Its molecular mass is approximately 68 kDa. LPO is a basic protein with an isoelectric point of 8.2. The carbohydrate content of this protein molecule is about 10% for the four glycosylation sites (Carlstrom, 1969). LPO contains a covalently linked prosthetic group in the catalytic centre which is a derivative of protoporphyrin IX (Thanabal & La Mar, 1989). The iron content of LPO is 0.07% (Paul & Ohlsson, 1985) corresponding to one iron atom per LPO molecule which is a part of the heme prosthetic group. The overall molecular structure of LPO is stabilized by a calcium ion which is strongly bound to LPO molecule through

LPO catalyzes a set of reactions where the resting ferric enzyme (Fe3+) is oxidized rapidly by hydrogen peroxide to form compound I (Kussendrager & van Hooijdonk, 2000), an oxyferryl porphyryl radical species where an oxygen is coupled by a double bond to the iron (Dolphin et

LPO + H2O2 Compound I + H2O (1)

Compound I + S Compound II + S\* (2)

Compound II + S LPO + S\* + H2O (3)

al., 1973) which subsequently oxidizes two aromatic substrate molecules as follows:

Where S is an aromatic substrate and S\* is an 1-electron oxidized form of substrate.

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

action of lactoperoxidase are harmless to mammalian cells (Reiter & Harnulv, 1984).

 ions) **(**Oram & Reiter, 1996; Hoogendoorn et al., 1977) at the expense of hydrogen peroxide (H2O2) in order to generate reactive products with a wide range of antimicrobial activities (Reiter & Harnulv, 1984; Reiter & Perraudin, 1991; Wolfson & Sumner, 1993). LPO also catalyzes the bielectronic oxidation (by two 1-electron steps) of a number of physiologically relevant aromatic organic compounds (Ciaccio et al., 2004; Zhang & Dunford 1993; Monzani et al., 1997; Metodiewa et al., 1989; Metodiewa et al., 1989; Ferrari et al., 1993; Doerge & Decker, 1994; Sipe, 1994; Cavalieri et al., 1997; Ghibaudi et al., 2000;

ion) or hypohalides (OI-

, OBr-

and

) to pseudohypohalide (OSCN-

or halides (I-

OCl-

, Br-

Ramakrishna et al., 1993).

seven-fold coordination.

**2.1 Mechanism of action** 

**3. Structure of lactoperoxidase** 

and Cl-

compounds are converted into useful antimicrobial products. Since LPO is able to bind and oxidize both of these compounds, the role of LPO in the treatment of TB appears to be quite plausible. It may be mentioned here that the peroxidase activity of *Mt*CP was shown to be associated with the activation of isoniazid (Zhang et al., 1992). It may also be mentioned here that the role of LPO has already been demonstrated in the bacterial clearance of airways by inhaling INH because LPO and H2O2 are present in the mucus of airways (Sawatdee et al., 2006). Thus, understanding the mode of binding of INH and PZA to LPO as well as the mechanisms of action of LPO with respect to these compounds will provide important insights on the possible mode of bindings of INH and PZA to bacterial enzymes *Mt*CP and PncA respectively.

**Isoniazid Isonicotinoyl radical**

Scheme 1.

**Pyrazinamide Pyrazinoic acid**

Scheme 2.

#### **2. Lactoperoxidase**

Lactoperoxidase (EC.1.11. 1.7) (LPO) belongs to the family of mammalian heme peroxidases which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). LPO is present in exocrine secretions such as milk, saliva and tears. Although it is produced at different sites in human body by various glands such as mammary, salivary and lachrymal with varying amino acid sequences but these were found to be chemically and immunologically similar (Kussendrager & van Hooijdonk, 2000). The

compounds are converted into useful antimicrobial products. Since LPO is able to bind and oxidize both of these compounds, the role of LPO in the treatment of TB appears to be quite plausible. It may be mentioned here that the peroxidase activity of *Mt*CP was shown to be associated with the activation of isoniazid (Zhang et al., 1992). It may also be mentioned here that the role of LPO has already been demonstrated in the bacterial clearance of airways by inhaling INH because LPO and H2O2 are present in the mucus of airways (Sawatdee et al., 2006). Thus, understanding the mode of binding of INH and PZA to LPO as well as the mechanisms of action of LPO with respect to these compounds will provide important insights on the possible mode of bindings of INH and PZA to bacterial enzymes

*Mt***CP / LPO**

**Isoniazid Isonicotinoyl radical**

**Pyrazinamide Pyrazinoic acid**

Lactoperoxidase (EC.1.11. 1.7) (LPO) belongs to the family of mammalian heme peroxidases which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). LPO is present in exocrine secretions such as milk, saliva and tears. Although it is produced at different sites in human body by various glands such as mammary, salivary and lachrymal with varying amino acid sequences but these were found to be chemically and immunologically similar (Kussendrager & van Hooijdonk, 2000). The

**PncA**

**N**

**N C**

**O**

**OH**

**C**

**O**

**N**

*Mt*CP and PncA respectively.

Scheme 1.

Scheme 2.

**2. Lactoperoxidase** 

**O NH**

**NH2**

**N**

**N C**

**O**

**NH2**

**N**

primary function of LPO is to catalyze the bielectronic oxidation of pseudohalide (SCN- ion) or halides (I- , Br and Cl- ) to pseudohypohalide (OSCN ion) or hypohalides (OI- , OBr and OCl ions) **(**Oram & Reiter, 1996; Hoogendoorn et al., 1977) at the expense of hydrogen peroxide (H2O2) in order to generate reactive products with a wide range of antimicrobial activities (Reiter & Harnulv, 1984; Reiter & Perraudin, 1991; Wolfson & Sumner, 1993). LPO also catalyzes the bielectronic oxidation (by two 1-electron steps) of a number of physiologically relevant aromatic organic compounds (Ciaccio et al., 2004; Zhang & Dunford 1993; Monzani et al., 1997; Metodiewa et al., 1989; Metodiewa et al., 1989; Ferrari et al., 1993; Doerge & Decker, 1994; Sipe, 1994; Cavalieri et al., 1997; Ghibaudi et al., 2000; Ramakrishna et al., 1993).

The biological significance of lactoperoxidase is related to its involvement in the natural host defense system against invading micro-organisms (Reiter & Harnulv, 1984; Reiter & Perraudin, 1991; Wolfson & Sumner, 1993). Apart from that, it was also reported to be involved in the antiviral activity (Mikola et al., 1995; Pourtois et al., 1990; Shin et al., 2005), degradation of various carcinogens and protection of animal cells against peroxidative effects (Tenovuo et al, 1985). It may be noted that the reaction products generated by the catalytic action of lactoperoxidase are harmless to mammalian cells (Reiter & Harnulv, 1984).

Lactoperoxidase is a heme-containing single chain protein with 595 amino acid residues. Its molecular mass is approximately 68 kDa. LPO is a basic protein with an isoelectric point of 8.2. The carbohydrate content of this protein molecule is about 10% for the four glycosylation sites (Carlstrom, 1969). LPO contains a covalently linked prosthetic group in the catalytic centre which is a derivative of protoporphyrin IX (Thanabal & La Mar, 1989). The iron content of LPO is 0.07% (Paul & Ohlsson, 1985) corresponding to one iron atom per LPO molecule which is a part of the heme prosthetic group. The overall molecular structure of LPO is stabilized by a calcium ion which is strongly bound to LPO molecule through seven-fold coordination.

#### **2.1 Mechanism of action**

LPO catalyzes a set of reactions where the resting ferric enzyme (Fe3+) is oxidized rapidly by hydrogen peroxide to form compound I (Kussendrager & van Hooijdonk, 2000), an oxyferryl porphyryl radical species where an oxygen is coupled by a double bond to the iron (Dolphin et al., 1973) which subsequently oxidizes two aromatic substrate molecules as follows:

LPO + H2O2 Compound I + H2O (1)

$$\text{Compound I} + \text{S} \to \text{Compound II} + \text{S}^\* \tag{2}$$

$$\text{Compound II} + \text{S} \rightarrow \text{LPO} + \text{S}^\* + \text{H}\_2\text{O} \tag{3}$$

Where S is an aromatic substrate and S\* is an 1-electron oxidized form of substrate.
