Identification of a New Gene Required for the Biosynthesis of Rhodoquinone in Rhodospirillum rubrum

Abstract

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 ...

The kynurenine pathway is essential for rhodoquinone biosynthesis in Caenorhabditis elegans

“…The biosynthesis of RQ in animals has remained a puzzle for decades (24). In bacteria and protists, RQ is derived from Q, and rquA is the gene signature for its biosynthesis (9,11). In contrast, in animals, RQ is not derived from Q, and no RQ-specific gene has been discovered.…”

“…The biosynthetic pathway of RQ has been extensively studied in Rhodospirillum rubrum. In this organism, RQ biosynthesis requires Q as a precursor (8). Subsequently, it was discovered that the R. rubrum rquA gene is essential for RQ, but not for Q biosynthesis (9).…”

“…In this organism, RQ biosynthesis requires Q as a precursor (8). Subsequently, it was discovered that the R. rubrum rquA gene is essential for RQ, but not for Q biosynthesis (9). Despite a comprehensive study of R. rubrum knock-out mutants, using aerobic versus anaerobic transcriptome data and comparative genomic analysis, no other genes besides rquA were identified to be essential for RQ biosynthesis (10).…”

Ancient Adaptive Lateral Gene Transfers in the Symbiotic Opalina–Blastocystis Stramenopile Lineage

“…Rhodoquinol is then reoxidized by the mitochondrial complex II catalyzing the reverse reaction as a fumarate reductase (van Hellemond and Tielens 1994;Tielens et al 2002). This pathway helps to produce ATP and to reduce the respiratory chain without using the mitochondrial complexes III to V. The putative methyltransferase RquA is required for rhodoquinone biosynthesis (Lonjers et al 2012) and its distribution among eukaryotes suggests that it is important for the adaptation of the mitochondrial metabolism to low-oxygen environments. In Blastocystis, RquA was suggested to be targeted to the MRO (Eme et al 2017).…”

Identification of the pathway of Rhodoquinone biosynthesis inC.elegans

“…The sole gene known to be required specifically for RQ synthesis and not for UQ synthesis is the 339 R.rubrum gene rquA (Lonjers et al, 2012). This is a methyltransferase that is related to the 340 quinone methyltransferases UbiG/COQ3 and UbiE/COQ5 that act in UQ synthesis ( molluscs, and annelids, the animal species known to make RQ, are different however -in 350 addition to COQ3 and COQ5, they encode an additional set of related quinone 351 methyltransferases (Fig 8a).…”