Kinetic studies on enoyl- ACP reductase from plasmodium falciparum : A potential target of antimalarials

Abstract

Enoyl-ACP reductase (FabI) catalyzes the rate-determining step in the type II fatty acid synthesis (FAS) pathway. This pathway is present in plants and bacteria. However, the recent demonstration of this pathway in the malaria-causing human parasite Plasmodium falciparum has opened new avenues for drug development targeting specific enzymes of this pathway (Surolia and Surolia, 2001).

Malaria is the most devastating of all apicomplexan-related human diseases. Despite enormous antimalarial efforts, the disease accounts for half a billion infections and three million deaths per year. Importantly, the presence of type I FAS in the human host makes drugs targeting type II FAS clinically suitable, as they would not inhibit the human counterpart.

Thesis Overview This thesis describes the successful cloning of fabI from P. falciparum and its expression in E. coli. The study aimed to gain insight into the structure and function of the enzyme and to better understand interactions between FabI, its substrates, and its well-known inhibitor, triclosan.

Triclosan: A broad-spectrum antibacterial effective against E. coli (Heath et al., 1998) and M. tuberculosis (McMurray et al., 1999).

Protein purification: Achieved by single-step affinity chromatography.

Characterization: Triclosan was identified as a slow, tight-binding inhibitor of FabI using UV spectrophotometry, fluorescence spectroscopy, and surface plasmon resonance (SPR).

Mutational analysis: Specific PfFabI residues were studied to determine their role in triclosan binding.

Key Findings Cloning and Expression:

fabI cloned via RT-PCR from parasite RNA into pET28a(+) vector.

Protein expressed with an N-terminal His-tag for purification.

A silent mutation corrected a stretch of adenines that caused premature transcription termination.

Enzyme Characterization:

PfFabI reduces enoyl-CoA substrates in vitro.

Kinetic constants (Km, Vmax) determined using crotonoyl-CoA and NADH.

PfFabI showed similarities to Brassica napus FabI but differences from E. coli FabI.

Inhibition by Triclosan:

Inhibition potentiated in the presence of NAD+.

Formation of a stable ternary complex (PfFabI-NAD+-triclosan) was key to inhibition.

SPR revealed a 300-fold increase in binding affinity in the presence of NAD+.

Mutational Analysis:

Mutants A217V, A217G, N218D, M281T, and F368I reduced triclosan affinity.

A217V caused a >7000-fold decrease in binding affinity compared to wild-type.

Broader Context Malaria remains a major global health challenge, with rising drug resistance.

Traditional antimalarials (quinine, chloroquine, mefloquine) have unclear mechanisms.

Many parasite biochemical pathways overlap with human pathways, limiting drug development.

Type II FAS enzymes like FabI and FabZ represent promising selective targets.

Publications from This Work Kapoor et al. (2001) - Kinetic determinants of enoyl-ACP reductase interaction with substrates and inhibitors.

Sharma et al. (2003) - Identification and inhibition of Plasmodium falciparum FabZ.

Kapoor et al. (2003) - Interactions of substrate with calreticulin.

Kapoor et al. (2004) - Mutational analysis of calreticulin carbohydrate-binding region.