Friday, November 22, 2024
11/22/2024
DESCRIPTION (provided by applicant): Rhodoquinone (RQ) is an essential cofactor used in the anaerobic energy metabolism of species such as the parasitic helminths, the free-living nematode Caenorhabditis elegans (C. elegans), and the purple non-sulfur bacterium, Rhodospirillum rubrum (R. rubrum). RQ is not synthesized or used in humans and other mammals with a primarily aerobic energy metabolism. However, RQ is structurally similar to ubiquinone (coenzyme Q or Q), an important lipid component involved in the aerobic respiratory chain. Both RQ and Q have a fully substituted benzoquinone ring and a polyisoprenoid side chain of varying length (depending on species). The only difference between the structures is that RQ has an amino group (NH2) instead of a methoxy group (OCH3) on the quinone ring. Therefore, the biosynthetic pathways of RQ and Q are proposed to be similar and may diverge from common precursors. The biosynthesis of Q has been well-characterized in both eukaryotic and prokaryotic species. It has recently been shown in my laboratory that the catabolism of Q is required for RQ biosynthesis in R. rubrum. A mutant strain (F11) of R. rubrum has also been identified which can synthesize Q, but not RQ, and therefore cannot grow anaerobically. The whole genomes of the mutant F11 and its spontaneous revertant RF110 were recently sequenced. The main focus of this proposal is to identify candidate gene(s) and polypeptide(s) responsible for RQ biosynthesis using the model organism R. rubrum. Selective inhibition of a unique enzyme target used in RQ biosynthesis (e.g. an aminotransferase) may lead to highly specific antihelminthic drugs that do not have a toxic effect on the host. There are three specific aims proposed for completion within the three-year award period. Specific Aim 1 involves the characterization of the conversion of Q to RQ in R. rubrum. Both in vivo and in vitro assays will be developed to identify new RQ biosynthetic intermediates, and the source of the amino group in RQ. Inhibition assays that target RQ biosynthesis will also be performed with commercially available aminotransferase inhibitors. Specific Aim 2 focuses on characterization of the F11 mutant using complementation experiments. From sequencing data, it was determined that the F11 mutation occurs in a putative methyltransferase gene. The corresponding gene product will be overexpressed and characterized. Specific Aim 3 will take advantage of bioinformatics to identify candidate aminotransferase and O-demethylase genes involved in RQ biosynthesis in R. rubrum. Deletion mutants will be prepared from the strongest candidates to determine their role in RQ biosynthesis. Unique RQ biosynthetic enzyme targets that are identified will ultimately be used for antihelminthic drug design. PUBLIC HEALTH RELEVANCE: Rhodoquinone (RQ) is an essential aminoquinone used in the anaerobic energy metabolism of the helminth parasites and it is not found or required in mammalian hosts. Characterization of genes and gene products in the RQ biosynthetic pathway in Rhodospirillum rubrum is the focus of this proposal, and may identify a parasite-specific target for drug development.
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