Using Lipoamidase as a Novel Probe To Interrogate the Importance of Lipoylation in Plasmodium falciparum

Lipoate is an essential cofactor for a small number of enzymes that are important for central metabolism. Malaria parasites require lipoate scavenged from the human host for growth and survival; however, it is not known why this cofactor is so important. To address this question, we designed a probe of lipoate activity based on the bacterial enzyme lipoamidase (Lpa). Expression of this probe in different subcellular locations allowed us to define the mitochondrion as the compartment housing essential lipoate metabolism. To gain further insight into the specific uses of lipoate in the mitochondrion, we designed a series of catalytically attenuated probes and employed the probes in conjunction with a chemical bypass system. These studies suggest that two lipoylated proteins are required for parasite survival. We were able to express Lpa with different catalytic abilities in different subcellular compartments and driven by different promoters, demonstrating the versatility of this tool and suggesting that it can be used as a probe of lipoate metabolism in other organisms. ABSTRACT Lipoate is a redox active cofactor that is covalently bound to key enzymes of oxidative metabolism. Plasmodium falciparum is auxotrophic for lipoate during the intraerythrocytic stages, but it is not known whether lipoate attachment to protein is required or whether attachment is required in a specific subcellular compartment of the parasite. To address these questions, we used an enzyme called lipoamidase (Lpa) as a probe of lipoate metabolism. Lpa was first described in Enterococcus faecalis, and it specifically cleaves protein-bound lipoate, inactivating enzymes requiring this cofactor. Enzymatically active Lpa could be expressed in the cytosol of P. falciparum without any effect on protein lipoylation or parasite growth. Similarly, Lpa could be expressed in the apicoplast, and although protein lipoylation was reduced, parasite growth was not inhibited. By contrast, while an inactive mutant of Lpa could be expressed in the mitochondrion, the active enzyme could not. We designed an attenuated mutant of Lpa and found that this enzyme could be expressed in the parasite mitochondrion, but only in conjunction with a chemical bypass system. These studies suggest that acetyl-CoA production and a cryptic function of the H protein are both required for parasite survival. Our study validates Lpa as a novel probe of metabolism that can be used in other systems and provides new insight into key aspects of mitochondrial metabolism that are responsible for lipoate auxotrophy in malaria parasites. IMPORTANCE Lipoate is an essential cofactor for a small number of enzymes that are important for central metabolism. Malaria parasites require lipoate scavenged from the human host for growth and survival; however, it is not known why this cofactor is so important. To address this question, we designed a probe of lipoate activity based on the bacterial enzyme lipoamidase (Lpa). Expression of this probe in different subcellular locations allowed us to define the mitochondrion as the compartment housing essential lipoate metabolism. To gain further insight into the specific uses of lipoate in the mitochondrion, we designed a series of catalytically attenuated probes and employed the probes in conjunction with a chemical bypass system. These studies suggest that two lipoylated proteins are required for parasite survival. We were able to express Lpa with different catalytic abilities in different subcellular compartments and driven by different promoters, demonstrating the versatility of this tool and suggesting that it can be used as a probe of lipoate metabolism in other organisms.