Rhodoquinone (RQ) is a required cofactor for anaerobic respiration in Rhodospirillum rubrum, and it is also found in several helminth parasites that utilize a fumarate reductase pathway. RQ is an aminoquinone that is structurally similar to ubiquinone (Q), a polyprenylated benzoquinone used in the aerobic respiratory chain. RQ is not found in humans or other mammals, and therefore, the inhibition of its biosynthesis may provide a novel antiparasitic drug target. To identify a gene specifically required for RQ biosynthesis, we determined the complete genome sequence of a mutant strain of R. rubrum (F11), which cannot grow anaerobically and does not synthesize RQ, and compared it with that of a spontaneous revertant (RF111). RF111 can grow anaerobically and has recovered the ability to synthesize RQ. The two strains differ by a single base pair, which causes a nonsense mutation in the putative methyltransferase gene rquA. To test whether this mutation is important for the F11 phenotype, the wildtype rquA gene was cloned into the pRK404E1 vector and conjugated into F11. Complementation of the anaerobic growth defect in F11 was observed, and liquid chromatography-time of flight mass spectrometry (LC-TOF-MS) analysis of lipid extracts confirmed that plasmid-complemented F11 was able to synthesize RQ. To further validate the requirement of rquA for RQ biosynthesis, we generated a deletion mutant from wild-type R. rubrum by the targeted replacement of rquA with a gentamicin resistance cassette. The ⌬rquA mutant exhibited the same phenotype as that of F11. These results are significant because rquA is the first gene to be discovered that is required for RQ biosynthesis. R hodoquinone (RQ) (Fig. 1, compound 1) is found in the mitochondrial membrane of parasitic helminths (43, 46) and other eukaryotic species capable of fumarate reduction, such as Euglena gracilis (17) and Caenorhabditis elegans (41). These species can adapt their metabolism to both aerobic and anaerobic conditions throughout their life cycle. Adult parasitic species such as Ascaris suum, Fasciola hepatica, and Haemonchus contortus rely heavily on fumarate reduction for their energy generation while inside a host organism, where the oxygen tension is very low (20,45,48). Under these conditions, the biosynthesis of RQ is upregulated; however, during free-living stages of their life cycle, the helminth parasites use primarily aerobic respiration, which requires ubiquinone (Q) (Fig. 1, compound 2) (20,44,48). The anaerobic energy metabolism of the helminths was reviewed previously (43,46). Humans and other mammalian hosts use Q for aerobic energy metabolism but do not produce or require RQ; therefore, the discovery of molecules that selectively inhibit RQ biosynthesis may lead to highly specific antihelminthic therapeutics that do not have a toxic effect on the host (24).The pathway of RQ biosynthesis has not been completely elucidated, and enzymes specifically required for RQ synthesis still must be identified. RQ is structurally similar to Q, an important ...