Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In normoxia they use ubiquinone (UQ), but in the anaerobic conditions inside the host, they require rhodoquinone (RQ) and greatly increase RQ levels. We previously showed the switch from UQ to RQ synthesis is driven by a change in substrates by the polyprenyltransferase COQ-2 (Del Borrello et al., 2019; Roberts Buceta et al., 2019) - how this substrate choice is made is unknown. Here, we show helminths make two coq-2 splice forms, coq-2a and coq-2e, and the coq-2e-specific exon is only found in species that make RQ. We show that in C. elegans COQ-2e is required for efficient RQ synthesis and for survival in cyanide. Crucially, parasites switch from COQ-2a to COQ-2e as they transition into anaerobic environments. We conclude helminths switch from UQ to RQ synthesis principally via changes in the alternative splicing of coq-2.

Terpenoid quinones are liposoluble redox-active compounds that serve as essential electron carriers and anti-oxidants. One such quinone, rhodoquinone (RQ), couples the respiratory electron transfer chain to the reduction of fumarate to facilitate anaerobic respiration. This mechanism allows RQ-synthesizing organisms to operate their respiratory chain using fumarate as a final electron acceptor. RQ biosynthesis is restricted to a handful of prokaryotic and eukaryotic organisms, and details of this biosynthetic pathway remain enigmatic. One gene, rquA, was discovered to be required for RQ biosynthesis in Rhodospirillum rubrum. However, the function of the gene product, RquA, has remained unclear. Here, using reverse genetics approaches, we demonstrate that RquA converts ubiquinone to RQ directly. We also demonstrate the first in vivo synthetic production of RQ in Escherichia coli and Saccharomyces cerevisiae, two organisms that do not natively produce RQ. These findings help clarify the complete RQ biosynthetic pathway in species which contain RquA homologs.

17Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In aerobic 18 environments they use ubiquinone (UQ) but in anaerobic environments inside the host, they require rhodoquinone 19 (RQ) and greatly increase RQ levels. The switch to RQ synthesis is driven by substrate selection by the 20 polyprenyltransferase COQ-2 but the mechanisms underlying this substrate choice are unknown. We found that 21 helminths make two coq-2 isoforms, coq-2a and coq-2e, by alternative splicing. COQ-2a is homologous to COQ2 22 from other eukaryotes but the COQ-2e-specific exon is only found in species that make RQ and its inclusion 23 changes the enzyme core. We show COQ-2e is required for RQ synthesis and for survival in cyanide in C. elegans. 24 Crucially, we see a switch from COQ-2a to COQ-2e as parasites transition into anaerobic environments. We 25 conclude that under anaerobic conditions helminths switch from UQ to RQ synthesis via alternative splicing of 26 coq-2. 27

Parasitic helminths infect more than 1.5 billion people, with third world countries being the most affected. Parasites are transmitted through soil and contaminated water and they feed on host tissue in order to grow and reproduce. Infections cause anemia, malabsorption of nutrients, and loss of appetite. While inside a host, parasites anaerobically respire by using an alternative electron transport chain that uses rhodoquinone (RQ) as an electron carrier instead of ubiquinone (Q), which is used in aerobic respiration. Mammalian hosts produce and use Q for aerobic respiration, but they do not make or require RQ. An ideal anti‐parasitic drug target would be an enzyme that is used only for the biosynthesis of RQ ‐ not the biosynthesis of Q. Caenorhabditis elegans have been used as a model system for parasitic helminths in order to investigate RQ biosynthesis. RNAi feeding experiments were performed to identify genes required for several steps in the RQ biosynthetic pathway in C. elegans. Several genes were identified that are common to both Q and RQ biosynthetic pathways (coq‐3, coq‐5, and coq‐6). However, knockdown of the kynu‐1 gene from the kynurenine pathway affected only RQ production. Additional experiments involving knock‐outs of other C. elegans genes in the kynurenine pathway confirmed the requirement of kynu‐1 and the corresponding arylamine products for the biosynthesis of RQ. The roles ofcoq‐2 and coq‐3 MV genes in the biosynthesis of RQ in C. elegans were further investigated in this study using RNAi knockdowns and characterization was performed with RT‐qPCR and LC‐MS. Support or Funding Information Gonzaga Science Research Program and Agencia Nacional para la Innovación y la Investigación ANII FCE_1_2014_104366

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