The kynurenine pathway is essential for rhodoquinone biosynthesis in Caenorhabditis elegans

Abstract

A key metabolic adaptation of some species that face hypoxia as part of their life cycle involves an alternative electron transport chain in which rhodoquinone (RQ) is required for fumarate reduction and ATP production. RQ biosynthesis in bacteria and protists requires ubiquinone (Q) as a precursor. In contrast, Q is not a precursor for RQ biosynthesis in animals such as parasitic helminths, and most details of this pathway have remained elusive. Here, we used Caenorhabditis elegans as a model animal to elucidate key steps in RQ biosynthesis. Using RNAi and a series of C. elegans mutants, we found that arylamine metabolites from the kynurenine pathway are essential precursors for RQ biosynthesis de novo. Deletion of kynu-1, encoding a kynureninase that converts l-kynurenine (KYN) to anthranilic acid (AA) and 3-hydroxykynurenine (3HKYN) to 3-hydroxyanthranilic acid (3HAA), completely abolished RQ biosynthesis but did not affect Q levels. Deletion of kmo-1, which encodes a kynurenine 3-monooxygenase that converts KYN to 3HKYN, drastically reduced RQ but not Q levels. Knockdown of the Q biosynthetic genes coq-5 and coq-6 affected both Q and RQ levels, indicating that both biosynthetic pathways share common enzymes. Our study reveals that two pathways for RQ biosynthesis have independently evolved. Unlike in bacteria, where amination is the last step in RQ biosynthesis, in worms the pathway begins with the arylamine precursor AA or 3HAA. Because RQ is absent in mammalian hosts of helminths, inhibition of RQ biosynthesis may have potential utility for targeting parasitic infections that cause important neglected tropical diseases.

Alternative splicing of coq-2 controls the levels of rhodoquinone in animals

“…The regulation of COQ-2 specificity by alternative splicing is thus a beautiful and rare example of this type of enzyme regulation by alternative splicing. We note that while alternative splicing of COQ-2 appears to be the key regulated step in determining RQ or UQ biosynthesis, we see no such splicing regulation for other enzymes that are required for both RQ and UQ synthesis downstream of COQ-2 ( Roberts Buceta et al, 2019 ), for example, there are no known splice variants of COQ-3 and COQ-5, which are quinone methylases downstream of COQ-2. This suggests that while COQ-2 can clearly discriminate between substrates that have/lack a 2-amino group, COQ-3 and COQ-5 would be more promiscuous than COQ-2 and act on both RQ and UQ precursors.…”

“…We previously showed that the key decision on whether to synthesize UQ to power aerobic metabolism or RQ to synthesize anaerobic metabolism is dictated by the choice of substrate of the polyprenyltransferase COQ-2 ( Del Borrello et al, 2019 ; Roberts Buceta et al, 2019 ). COQ-2 must switch from using 4HB to synthesize UQ in aerobic conditions to 3HA to synthesize RQ in anaerobic conditions.…”

“…Lipid extractions of C. elegans N2 and mutant strains were performed on ~100 mg worm pellets after adding 1000 pmol UQ 3 internal standard ( Roberts Buceta et al, 2019 ). LC-MS samples were prepared from lipid extracts and diluted 1:100 ( Bernert et al, 2019 ).…”

“…Among animals, RQ has only been described in nematodes, platyhelminths, mollusks, and annelids ( Van Hellemond et al, 1995 ). The key steps of RQ biosynthesis in animals were recently elucidated ( Roberts Buceta et al, 2019 ; Del Borrello et al, 2019 ). In contrast to bacteria, where RQ derives from UQ ( Bernert et al, 2019 ; Brajcich et al, 2010 ), RQ biosynthesis in animals requires precursors derived from tryptophan ( Figure 1B ).…”

“…Recent studies performed on C. elegans have demonstrated that animals that lack a functional kynureninase pathway (e.g. strains carrying mutations in kynu-1, the sole kynureninase) are unable to synthesize RQ ( Del Borrello et al, 2019 ; Roberts Buceta et al, 2019 ). It is presumed that 3-hydroxyanthranilic acid (3HA, also sometimes referred to as 3HAA) from the kynurenine pathway is prenylated in a reaction catalyzed by COQ-2.…”