CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW
1.13 Apicoplast Type II FAS-mediated chain elongation
Figure 1.7 illustrates the proposed reaction steps used by apicomplexa to produce palmitic acid (Ralph et al., 2004b; Surolia et al., 2004; Wilson, 2002). Malonyl-CoA is attached to acyl carrier protein (ACP) in a ping-pong reaction mechanism catalysed by malonyl-CoA:ACP transacylase.
This enzyme has been cloned, expressed and characterised in P. falciparum (Prigge et al., 2003).
Although this enzyme is necessary for parasite survival, its potential as an anti-parasitic agent is yet to be exploited (Surolia et al., 2004). Malonyl-ACP is the substrate that is continually added to the fatty acid chain, thus each cycle results in the addition of two carbon moieties from malonyl-ACP. The chain elongation step is divided into four distinct reaction steps namely:
condensation, reduction, dehydration and reduction (Surolia et al., 2004).
The Type II FAS-catalysed chain elongation in the apicomplexa plastid begins with the condensation of acetyl-CoA with malonyl-ACP; unlike the Type I FAS chain elongation that uses acetyl-ACP as substrate for the condensation step (Surolia et al., 2004; Wilson, 2002).This first step is catalysed by β-ketoacyl-ACP synthase III (KAS III or FabH), while the subsequent condensation steps (cycles 2 – 7) are catalysed by a FabB/F (KAS I/II) isoezyme (Surolia et al., 2004; Wilson, 2002). FabH is annotated in P. falciparum (Plasmodb ID PFB0505c, located on chromosome 2), vivax, knowlesi, berghei and chabaudi as a beta-ketoacyl-ACP synthase III precursor. FabB/F is annotated in P. falciparum (Plasmodb ID PFF1275c, located on chromosome 6), vivax, knowlesi, berghei, yoelii and chabaudi as the beta-ketoacyl-ACP synthase III precursor. P. falciparum FabH is inhibited by 1,2, dithiole-3-one (an analogue of thiolactomycin) with an IC50 of 0.5 – 10 µM (Prigge et al., 2003). Thiolactomycin (Figure 1.8) is
a competitive inhibitor of bacterial and plant ketoacyl-ACP synthase (Sasaki and Nagano, 2004;
Surolia et al., 2004) and has been shown to inhibit P. falciparum growth in human erythrocyte at concentrations less than 50 µM (Gornicki, 2003).
The second step in the Type II FAS chain elongation is the reduction of β-ketoacyl-ACP to β- hydroxyacyl-ACP. This reaction step is catalysed by NADP-dependent β-ketoacyl-ACP reductase (Surolia et al., 2004). This enzyme is annotated in P. falciparum as putative 3-oxoacyl- ACP reductase (Plasmodb ID: PFI1125c, located on chromosome 9), as well as in vivax, knowlesi, berghei, yoelii and chabaudi (Gornicki, 2003). No inhibitor of this enzyme has been identified. β-hydroxyacyl-ACP is dehydrated to enoyl-ACP by β-hydroxyacyl-ACP dehydrase (FabA/Z). The FabA isoenzyme is bifunctional, it catalyses the dehydration of β-hydroxyacyl- ACP and the trans-isomerisation of trans-enoyl-ACP to cis-enoyl-ACP. Cis-enoyl-ACP is channelled into the production of unsaturated fatty acids (Surolia et al., 2004; Surolia and Surolia, 2001). FabZ is characterised in P. falciparum (Plasmodb ID PF13_0128, located on chromosome 13) (Sharma et al., 2003). Orthologues of FabZ have been identified in P. vivax, knowlesi, berghei, yoelii and chabaudi; and annotated as the β-hydroxyacyl-ACP dehydrase precursor in the Plasmodb database. In the final elongation step, enoyl-ACP is reduced to butyryl-ACP by enoyl-ACP redcutase (FabI). This enzyme is NADH-dependent in apicomplexa Type II FAS as contrasting the NADPH-dependent homologue of the Type I FAS (Surolia et al., 2004; Surolia and Surolia, 2001). FabI is inhibited by triclosan (Figure 1.8) a broad spectrum antimicrobial agent (Suguna et al., 2001; Surolia et al., 2004; Surolia and Surolia, 2001). In Plasmodium, triclosan inhibits FabI (Plasmodb ID PFF0730c, located on Chromosome 6) with an IC50 of 0.2 – 1.2 µM. The FabI ortholgue has been annotated in P. vaivax, knowlesi, berghei, yoelii and chabaudi. The final product of the first elongation cycle (butyryl-ACP) enters the second cycle by condensing with malonyl-ACP catalysed by FabB/F. Cerulenin (Figure 1.8) is an inhibitor of FabB/F (Johnson et al., 1994; Lack et al., 2006). After the seventh cycle, the resulting product is a 16-carbon atom fatty acid called palmitioyl-ACP. This product is hydrolysed to palmitate (Surolia et al., 2004; Wilson, 2002).
In summary, fatty acid metabolism is vital to energy and membrane biogenesis. Acetyl-CoA carboxylase is central to de novo fatty acid metabolism. The two major types of acetyl-CoA
carboxylase are the multi-enzyme domain type found in humans, yeast and monocot plants and the multi-subunit enzyme complex found in bacteria and dicot plants. De novo fatty acid synthesis is initiated by the acetyl-CoA carboxylase-mediated carboxylation of acetyl-CoA to form malonyl-CoA. This reaction is the committed step in fatty acid synthesis and is ATP- dependent. Malonyl-CoA is elongated to palmitic acid via a series of repetitive steps of condensation, reduction and dehydration reactions catalysed by fatty acid synthase (FAS).
Figure 1.7 Type II fatty acid synthase-mediated synthesis of palmitate. Reaction products and intermediates are in blue and inhibitors are in magenta. Squiggle arrows indicate a multi-
S O
CoA O
O O
S CoA
S O
CoA O
O O
S ACP
O
S ACP
O
O
S ACP
OH
O
S ACP
ACP S O
CO2 CoASH + HCO3 ADP
Pi H+
S O
ACP
NADH+H+
NAD+
H2O NADPH+H+
NADP+
ACP
CH3 [CH2]14 C O
S ACP
CH3 [CH2]14 C
O OH
H2O ACP
_
ACP
CoASH
O
O O
S ACP
CO2
Acetyl-CoA carboxylase
Malonyl-CoA:ACP transacylase FabD
Ketoacyl-ACP synthase III FabH
Ketoacyl-ACP reductase FabG
Hydroxyacyl-ACP dehydrase FabZ/A
Enoyl-ACP reductase FabI
Cycle 2
5 cycles
Hydrolysis
Acetyl-CoA Malonyl-CoA
Malonyl-ACP
Acetyl-CoA
Ketoacyl-ACP
Hydroxyacyl-ACP
Enoyl-ACP Butyryl-ACP
Malonyl-ACP
Palmitoyl-ACP Acyl(C6)-ACP
Palmitate Ketoacyl-ACP synthase III
FabB/F
Thiolactomycin
Triclosan Cerulenin Fenoxaprop
Clodinafop
_
_
_
There are two types of FAS responsible for elongation of malonyl-CoA, namely the Type I FAS and Type II FAS. The type I FAS is a large multi-enzyme domain (multi-functional) polypeptide found in humans and fungi, while the Type II FAS is made up of discrete and mono-functional enzymes (complex) found in bacteria and plant chloroplasts. Apicomplexan parasites have been shown to express a nucleus-encoded acetyl-CoA carboxylase and Type II FAS, which are targeted to the apicoplast. However, Cryptosporidium parvum (an apicomplexa) has been shown to express cytoplasmic-targeted acetyl-CoA carboxylase and Type I FAS (Zhu, 2004; Zhu et al., 2004; Zhu et al., 2000). There are inhibitors of acetyl-CoA carboxylase and FAS. Fatty acid metabolism is targeted for anti-parasite chemotherapy due to the relevance of fatty acid metabolism to the survival of parasites.
O
N O
O C COOH
H CH3 Cl
O
O C COOH
H CH3 Cl
Cl
O
OH Cl
Cl Cl
O
O NH2
O CH3 S
CH3 OH
O
O O
CH2CH2CH3
N O
CH2CH3 CH3CH2SCHCH2
CH3
Sethoxydim
Fenoxaprop (Fop) Diclofop (Fop)
Triclosan
Thiolactomycin Cerulenin
Figure 1.8 Inhibitors of acetyl-CoA carboxylase and Type II fatty acid synthase. (Jelenska et al., 2001; Jelenska et al., 2002; Surolia et al., 2004)